Unique associated kaposi&#39;s sarcoma virus sequences and uses thereof

ABSTRACT

This invention provides an isolated peptide encoded by a nucleic acid which is at least 30 nucleotides in length and has a sequence which uniquely defines a herpesvirus associated with Kaposis&#39; sarcoma, which herpesvirus is present in and recoverable from the HBL-6 cell line (ATCC Accession No. CRL 11762).

This application is a divisional of U.S. Ser. No. 11/801,641, filed May9, 2007, which is a continuation of U.S. Ser. No. 09/607,179, filed Jun.29, 2000, which is a continuation of U.S. Ser. No. 08/793,624, filedFeb. 18, 1997, now U.S. Pat. No. 6,150,093, issued Nov. 21, 2000, whichis a 5371 national stage application of PCT International ApplicationNo. PCT/US95/10194, filed Aug. 11, 1995, which is a continuation-in-partof U.S. Ser. No. 08/420,235, filed Apr. 11, 1995, now U.S. Pat. No.5,801,042, issued Sep. 1, 1998, which is a continuation-in-part of U.S.Ser. No. 08/343,101, filed Nov. 21, 1994, now U.S. Pat. No. 5,830,759,issued Nov. 3, 1998, which is a continuation-in-part of U.S. Ser. No.08/292,365, filed Aug. 18, 1994, now abandoned, the contents all ofwhich are hereby incorporated by reference in their entireties into thisapplication.

The invention disclosed herein was made with Government support under aco-operative agreement CCU210852 from the Centers for Disease Controland Prevention, of the Department of Health and Human Services.Accordingly, the U.S. Government has certain rights in this invention.

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“110426_(—)0575_(—)45185-CAAZ-PCT-US_SubSequenceListingAHC.txt” which is122 kilobytes in size, and which was created Apr. 26, 2011, in theIBM-PCT machine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Apr. 26, 2011 as apart of this application.

Throughout this application, various publications may be referenced byArabic numerals in brackets. Full citations for these publications maybe found at the end of each Experimental Details Section. Thedisclosures of the publications cited herein are in their entiretyhereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Kaposi's sarcoma (KS) is the most common neoplasm occurring in personswith acquired immunodeficiency syndrome (AIDS). Approximately 15-20% ofAIDS patients develop this neoplasm which rarely occurs inimmunocompetent individuals [13, 14]. Epidemiologic evidence suggeststhat AIDS-associated KS (AIDS-KS) has an infectious etiology. Gay andbisexual AIDS patients are approximately twenty times more likely thanhemophiliac AIDS patients to develop KS, and KS may be associated withspecific sexual practices among gay men with AIDS [6, 15, 55, 83]. KS isuncommon among adult AIDS patients infected through heterosexual orparenteral HIV transmission, or among pediatric AIDS patients infectedthrough vertical HIV transmission [77]. Agents previously suspected ofcausing KS include cytomegalovirus, hepatitis B virus, humanpapillomavirus, Epstein-Barr virus, human herpesvirus 6, humanimmunodeficiency virus (HIV), and Mycoplasma penetrans [18, 23, 85, 91,92]. Non-infectious environmental agents, such as nitrite inhalants,also have been proposed to play a role in KS tumorigenesis [33].Extensive investigations, however, have not demonstrated an etiologicassociation between any of these agents and AIDS-KS [37, 44, 46, 90].

SUMMARY OF THE INVENTION

This invention provides an isolated DNA molecule which is at least 30nucleotides in length and which uniquely defines a herpesvirusassociated with Kaposi's sarcoma. This invention provides an isolatedherpesvirus associated with Kaposi's sarcoma.

This invention provides a method of vaccinating a subject for KS,prophylaxis diagnosing or treating a subject with KS and detectingexpression of a DNA virus associated with Kaposi's sarcoma in a cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1:

Agarose gel electrophoresis of RDA products from AIDS-KS tissue anduninvolved tissue. RDA was performed on DNA extracted from KS skintissue and uninvolved normal skin tissue obtained at autopsy from ahomosexual man with AIDS-KS. Lane 1 shows the initial PCR amplifiedgenomic representation of the AIDS-KS DNA after Bam HI digestion. Lanes2-4 show that subsequent cycles of ligation, amplification,hybridization and digestion of the RDA products resulted inamplification of discrete bands at 380, 450, 540 and 680 bp. RDA of theextracted AIDS-KS DNA performed against itself resulted in a single bandat 540 bp (lane 5). Bands at 380 bp and 680 bp correspond to KS330Bamand KS627Bam respectively after removal of 28 bp priming sequences.Bands at 450 and 540 bp hybridized nonspecifically to both KS and non-KShuman DNA. Lane M is a molecular weight marker.

FIGS. 2A-2B:

Hybridization of ³²P-labelled KS330Bam (FIG. 2A) and KS627Bam (FIG. 2B)sequences to a representative panel of 19 DNA samples extracted from KSlesions and digested with Bam HI. KS330Bam hybridized to 11 of the 19and KS627Bam hybridized to 12 of the 19 DNA samples from AIDS-KSlesions. Two additional cases (lanes 12 and 13) were shown to have faintbands with both KS330Bam and KS627Bam probes after longer exposure. Onenegative specimen (lane 3) did not have microscopically detectable KS inthe tissue specimen. Seven of 8 additional KS DNA samples alsohybridized to both sequences.

FIGS. 3A-3F:

Nucleotide sequences of the DNA herpesvirus associated with KS (KSHV).(SEQ ID NOs: 1, 36-40, respectively).

FIGS. 4A-4B:

PCR amplification of a representative set of KS-derived DNA samplesusing KS330₂₃₄ primers. FIG. 4A shows the agarose gel of theamplification products from 19 KS DNA samples (lanes 1-19) and FIG. 4B,shows specific hybridization of the PCR products to a ³²P end-labelled25 bp internal oligonucleotide (FIG. 3B) after transfer of the gel to anitrocellulose filter. Negative samples in lanes 3 and 15 respectivelylacked microscopically detectable KS in the sample or did not amplifythe constitutive p53 exon 6, suggesting that these samples were negativefor technical reasons. An additional 8 AIDS-KS samples were amplifiedand all were positive for KS330₂₃₄. Lane 20 is a negative control andLane M is a molecular weight marker.

FIG. 5:

Southern blot hybridization of KS330Bam and KS627Bam to AIDS-KS genomicDNA extracted from three subjects (lanes 1, 2, and 3) and digested withPvuII. Based on sequence information (FIG. 3A), restricted sites for PvuII occur between by 12361-12362 of the KSHV sequence (FIG. 3A, SEQ IDNO: 1), at bp 134 in KS330Bam (FIG. 3B, SEQ ID NO: 36) and bp 414 inKS627Bam (FIG. 3C, SEQ ID NO: 37). KS330Bam and KS627Bam failed tohybridize to the same fragments in the digests indicating that the twosequences are separated from each other by one or more intervening BamHI restriction fragments. Digestion with Pvu II and hybridization toKS330Bam resulted in two distinct banding patterns (lanes 1 and 2 vs.lane 3) suggesting variation between KS samples.

FIG. 6:

Comparison of amino acid homologies between EBV ORF BDLF1 (SEQ IDNO:47), HSVSA ORF 26 (SEQ ID NO:46) and a 918 bp reading frame of theKaposi's sarcoma agent which includes KS330Bam (SEQ ID NO:25). Aminoacid identity is denoted by reverse lettering. In HSVSA, ORF 26 encodesa minor capsid VP23 which is a late gene product.

FIG. 7:

Subculture of Raji cells co-cultivated with BCBL-1 cells treated withTPA for 2 days. PCR shows that Raji cells are positive for KSHVsequences and indicate that the agent is a transmissible virus.

FIG. 8:

A schematic diagram of the orientation of KSHV open reading framesidentified on the KS5 20,710 bp DNA fragment. Homologs to each openreading frame from a corresponding region of the herpesvirus saimiri(HSVSA) genome are present in an identical orientation, except for theregion corresponding to the ORF 28 of HSVSA (middle schematic section).The shading for each open reading frame corresponds to the approximate %amino acid identity for the KSHV ORF compared to this homolog in HSVSA.Noteworthy homologs that are present in this section of DNA includehomologs to thymidine kinase (ORF21), gH glycoprotein (ORF22), majorcapsid protein (ORF25) and the VP23 protein (ORF26) which contains theoriginal KS330Bam sequence derived by representational differenceanalysis.

FIG. 9:

The ˜200 kD antigen band appearing on a Western blot of KS patient seraagainst BCBL1 lysate (B1) and Raji lysate (RA). M is molecular weightmarker. The antigen is a doublet between ca. 210 kD and 240 kD.

FIG. 10:

5 control patient sera without KS (A1N, A2N, A3N, A4N and A5N). B1=BCBL1lysate, RA=Raji lysate. The 220 kD band is absent from the Western blotsusing patient sera without KS.

FIG. 11:

In this figure, 0.5 ml aliquots of the gradient have been fractionated(fractions 1-62) with the 30% gradient fraction being at fraction No. 1and the 10% gradient fraction being at fraction No. 62. Each fractionhas been dot hybridized to a nitrocellulose membrane and then a³²P-labeled KSHV DNA fragment, KS631Bam has been hybridized to themembrane using standard techniques. The figure shows that the majorsolubilized fraction of the KSHV genome bands (i.e. is isolated) infractions 42 through 48 of the gradient with a high concentration of thegenome being present in fraction 44. A second band of solubilized KSHVDNA occurs in fractions 26 through 32.

FIG. 12:

Location, feature, and relative homologies of KS5 open reading framescompared to translation products of herpesvirus saimiri (HSV), equineherpesvirus 2 (EHV2) and Epstein-Barr virus (EDV).

FIG. 13:

Indirect immunofluorescence end-point and geometric mean titers (GMT) inAIDS-KS and AIDS control sera against HBL-6 and P3H3 prior to and afteradsorption with P3H3.

FIG. 14:

Genetic map of KS5, a 20.7 kb lambda phage clone insert derived from ahuman genomic library prepared from an AIDS-KS lesion. Seventeen partialand complete open reading frames (ORFs) are identified with arrowsdenoting reading frame orientations. Comparable regions of theEpstein-Barr virus (EBV) and herpesvirus saimiri (HVS) genomes are shownfor comparison. Levels of amino acid similarity between KSHV ORFs areindicated by shading of EBV and HVS ORFs (black, over 70% similarity;dark gray, 55-70% similarity; light gray, 40-54% similarity; white, nodetectable homology). Domains of conserved herpesvirus sequence blocksand locations of restriction endonuclease sites used in subcloning areshown beneath the KSHV map (B, Bam HI site; N, Not I site). The smallBam HI fragment (black) in the VP23 gene homolog corresponds to theKS330Bam fragment generated by representational difference analysiswhich was used to identify the KS5 lambda phage clone.

FIGS. 15A-15B:

Phylogenetic trees of KSHV based on comparison of aligned amino acidsequences between herpesviruses for the MCP gene and for a concatenatednine-gene set. The comparison of MCP sequences (FIG. 15A) was obtainedby the neighbor-joining method and is shown in unrooted form with branchlengths proportional to divergence (mean number of substitution eventsper site) between the nodes bounding each branch. Comparable resultswere obtained by maximum parsimony analysis. The number of times out of100 bootstrap samplings the division indicated by each internal branchwas obtained are shown next to each branch; bootstrap values below 75are not shown. FIG. 15B is a phylogenetic tree of gammaherpesvirussequences based on a nine-gene set CS1 (see text) and demonstrates thatKSHV is most closely related to the gamma-2 herpesvirus sublineage,genus Rhadinovirus. The CS1 amino acid sequence was used to infer a treeby the Protml maximum likelihood method; comparable results, not shownwere obtained with the neighbor-joining and maximum parsimony methods.The bootstrap value for the central branch is marked. On the basis ofthe MCP analysis, the root must lie between EBV and the other threespecies. Abbreviations for virus species used in the sequencecomparisons are 1) Alphaherpesvirinae: HSV1 and HSV2, herpes simplexvirus types 1 and 2; EHV1, equine herpesvirus 1; PRV, pseudorabiesvirus; and VZV, varicella-zoster virus, 2) Betaherpesvirinae: HCMV,human cytomegalovirus; HHV6 and HHV7, human herpesviruses 6 and 7, and3) Gammaherpesvirinae: HVS, herpesvirus saimiri; EHV2, equineherpesvirus 2; EBV, Epstein-Barr virus; and Kaposi's sarcoma-associatedherpesvirus.

FIGS. 16A-16B:

CHEF gel electrophoresis of BCBL-1 DNA hybridized to KS631Bam (FIG. 16A)and EBV terminal repeat (FIG. 16B). KS631Bam hybridizes to a band at 270kb as well as to a diffuse band at the origin. The EBV termini sequencehybridizes to a 150-160 kb band consistent with the linear form of thegenome. Both KS631Bam (dark arrow) and an EBV terminal sequencehybridize to high molecular weight bands immediately below the originindicating possible concatemeric or circular DNA. The high molecularweight KS631Bam hybridizing band reproduces poorly but is visible on theoriginal autoradiographs.

FIG. 17:

Induction of KSHV and EBV replication in BCBL-1 with increasingconcentrations of TPA. Each determination was made in triplicate after48 h of TPA incubation and hybridization was standardized to the amountof cellular DNA by hybridization to beta-actin. The figure shows themean and range of relative increase in hybridizing genome for EBV andKSHV induced by TPA compared to uninduced BCBL-1. TPA at 20 ng/mlinduced an eight-fold increase in EBV genome (upper line) at 48 hcompared to only a 1.4 fold increase in KSHV genome (lower line).Despite the lower level of KSHV induction, increased replication of KSHVgenome after induction with TPA concentrations over 10 ng/ml wasreproducibly detected.

FIGS. 18A-18C:

In situ hybridization with an ORF26 oligomer to BCBL-1, Raji and RCC-1cells. Hybridization occurred to nuclei of KSHV infected BCBL-1 (FIG.18A), but not to uninfected Raji cells (FIG. 18B). RCC-1, a Raji cellline derived by cultivation of Raji with BCBL-1 in communicatingchambers separated by a 0.4 5μ filter, shows rare cells with positivehybridization to the KSHV ORF26 probe (FIG. 18C).

FIGS. 19A-19D:

Representative example of IFA staining of HBL-6 with AIDS-KS patientsera and control sera from HIV-infected patients without KS. BothAIDS-KS (FIG. 19A) and control (FIG. 19B) sera show homogeneous stainingof HBL-6 at 1:50 dilution. After adsorption with paraformaldehyde-fixedP3H3 to remove cross-reacting antibodies directed against lymphocyte andEBV antigens, antibodies from AIDS-KS sera localize to HBL-6 nuclei(FIG. 19C). P3H3 adsorption of control sera eliminates immunofluorescentstaining of HBL-6).

FIGS. 20A-20B:

Longitudinal PCR examination for KSHV DNA of paired PBMC samples fromAIDS-KS patients (A) and homosexual/bisexual AIDS patients without KS(B). Time 0 is the date of KS onset for cases or other AIDS-definingillness for controls. All samples were randomized and examined blindly.Overall, 7 of the KS patients were KSHV positive at both examinationdates (solid bars) and 5 converted from a negative to positive PBMCsample (forward striped bars) immediately prior to or after KS onset.Two previously positive KS patients were negative after KS diagnosis(reverse striped bars) and the remaining KS patients were negative atboth timepoints (open bars). Two KS converted from negative to positiveand one control patient reverted from PCR positive to negative for KSHVDNA.

FIG. 21:

Sample collection characteristics for AIDS-KS patients, gay/bisexualAIDS patients and hemophilic AIDS patients.

FIG. 22:

PCR analysis of KS330₂₃₃ in DNA samples from patients with Kaposi'ssarcoma and tumor controls.

FIG. 23:

Characteristics of the study population of patients with KS and withoutKS.

FIG. 24:

Prevalence of antibody to KSHV p40 in HIV-1 positive patients with andwithout KS.

FIG. 25:

Comparison of KS patients with and without antibody to KSHV p40.

FIG. 26:

Prevalence of antibody detectable by indirect immunofluorescence to KSHVantigens in chemically induced BCBL-1 cells in HIV-1 positive patientswith and without KS.

FIGS. 27A-27B:

Specific recognition of KSHV polypeptides in chemically treated BCBL-1cells. FIG. 27A shows reactivity of untreated BCBL-1 and B95-8 cellswith RM, a reference human antibody to EBV. RM recognizes the EBVpolypeptides EBNA1 and p21 in the BCBL-1 cells. FIG. 27B showsreactivity of untreated and chemically treated cells with serum 01-03from a patient with KS. Cells were treated with TPA and n-butyrate for48 hrs. For description of the cell lines see Materials and Methods. Theimmunoblots were prepared from 10% SDS polyacrylamide gels.

FIGS. 28A-28D:

Detection of KSHV p40 by sera from patients with KS. Extracts wereprepared from BCBL-1 cells (containing KSHV and EBV) and Clone HH514-16cells (containing only EBV) that were uninduced or treated for 48 hrswith chemical inducing agents, n-butyrate, TPA, or a combination of thetwo chemicals. Immunoblots prepared from 12% SDS polyacrylamide gelswere reacted with a 1:200 dilution of serum from HIV-1 positivepatients. FIG. 28A shows serum 01-06 from a patient with KS. FIG. 28Bshows serum 01-07 from a patient without KS. FIG. 28C shows serum 04-01from a patient with KS. FIG. 28D shows serum 01-03 from a patient withKS.

FIGS. 29A-29F:

Detection of KSHV lytic cycle antigens by indirect immunofluorescence.BCBL-1 cells were untreated (FIGS. 29A, 29C, and 29E) or treated withn-butyrate (FIGS. 29B, 29D, and 29F) for 48 hrs. Indirectimmunofluorescence with a 1:10 dilution of serum from two patients withKS, 04-18 (FIGS. 29A, and 29B) and 04-38 (FIGS. 29E, and 29F) and aserum, 04-37 (FIGS. 29C, and 29D), from a patient without KS.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following standard abbreviations are used throughout thespecification to indicate specific nucleotides:

-   -   C=cytosine A=adenosine    -   T=thymidine G=guanosine

The term “nucleic acids”, as used herein, refers to either DNA or RNA.“Nucleic acid sequence” or “polynucleotide sequence” refers to a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotidebases read from the 5′ to the 3′ end. It includes both self-replicatingplasmids, infectious polymers of DNA or RNA and nonfunctional DNA orRNA.

By a nucleic acid sequence “homologous to” or “complementary to”, it ismeant a nucleic acid that selectively hybridizes, duplexes or binds toviral DNA sequences encoding proteins or portions thereof when the DNAsequences encoding the viral protein are present in a human genomic orcDNA library. A DNA sequence which is homologous to a target sequencecan include sequences which are shorter or longer than the targetsequence so long as they meet the functional test set forth.Hybridization conditions are specified along with the source of the CDNAlibrary.

Typically, the hybridization is done in a Southern blot protocol using a0.2×SSC, 0.1% SDS, 65° C. wash. The term “SSC” refers to acitrate-saline solution of 0.15 M sodium chloride and 20 Mm sodiumcitrate. Solutions are often expressed as multiples or fractions of thisconcentration. For example, 6×SSC refers to a solution having a sodiumchloride and sodium citrate concentration of 6 times this amount or 0.9M sodium chloride and 120 mM sodium citrate. 0.2×SSC refers to asolution 0.2 times the SSC concentration or 0.03 M sodium chloride and 4mM sodium citrate.

The phrase “nucleic acid molecule encoding” refers to a nucleic acidmolecule which directs the expression of a specific protein or peptide.The nucleic acid sequences include both the DNA strand sequence that istranscribed into RNA and the RNA sequence that is translated intoprotein. The nucleic acid molecule include both the full length nucleicacid sequences as well as non-full length sequences derived from thefull length protein. It being further understood that the sequenceincludes the degenerate codons of the native sequence or sequences whichmay be introduced to provide codon preference in a specific host cell.

The phrase “expression cassette”, refers to nucleotide sequences whichare capable of affecting expression of a structural gene in hostscompatible with such sequences. Such cassettes include at leastpromoters and optionally, transcription termination signals. Additionalfactors necessary or helpful in effecting expression may also be used asdescribed herein.

The term “operably linked” as used herein refers to linkage of apromoter upstream from a DNA sequence such that the promoter mediatestranscription of the DNA sequence.

The term “vector”, refers to viral expression systems, autonomousself-replicating circular DNA (plasmids), and includes both expressionand nonexpression plasmids. Where a recombinant microorganism or cellculture is described as hosting an “expression vector,” this includesboth extrachromosomal circular DNA and DNA that has been incorporatedinto the host chromosome(s). Where a vector is being maintained by ahost cell, the vector may either be stably replicated by the cellsduring mitosis as an autonomous structure, or is incorporated within thehost's genome.

The term “plasmid” refers to an autonomous circular DNA molecule capableof replication in a cell, and includes both the expression andnonexpression types. Where a recombinant microorganism or cell cultureis described as hosting an “expression plasmid”, this includes latentviral DNA integrated into the host chromosome(s). Where a plasmid isbeing maintained by a host cell, the plasmid is either being stablyreplicated by the cells during mitosis as an autonomous structure or isincorporated within the host's genome.

The phrase “recombinant protein” or “recombinantly produced protein”refers to a peptide or protein produced using non-native cells that donot have an endogenous copy of DNA able to express the protein. Thecells produce the protein because they have been genetically altered bythe introduction of the appropriate nucleic acid sequence. Therecombinant protein will not be found in association with proteins andother subcellular components normally associated with the cellsproducing the protein.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acid molecules or polynucleotides:“reference sequence”, “comparison window”, “sequence identity”,“percentage of sequence identity”, and “substantial identity”. A“reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing or may comprise a complete cDNA orgene sequence.

Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethod of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA)85:2444, or by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).

As applied to polypeptides, the terms “substantial identity” or“substantial sequence identity” mean that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap which share at least 90 percent sequence identity, preferably atleast 95 percent sequence identity, more preferably at least 99 percentsequence identity or more.

“Percentage amino acid identity” or “percentage amino acid sequenceidentity” refers to a comparison of the amino acids of two polypeptideswhich, when optimally aligned, have approximately the designatedpercentage of the same amino acids. For example, “95% amino acididentity” refers to a comparison of the amino acids of two polypeptideswhich when optimally aligned have 95% amino acid identity. Preferably,residue positions which are not identical differ by conservative aminoacid substitutions. For example, the substitution of amino acids havingsimilar chemical properties such as charge or polarity are not likely toeffect the properties of a protein. Examples include glutamine forasparagine or glutamic acid for aspartic acid.

The phrase “substantially purified” or “isolated” when referring to aherpesvirus peptide or protein, means a chemical composition which isessentially free of other cellular components. It is preferably in ahomogeneous state although it can be in either a dry or aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. Generally, a substantially purified or isolatedprotein will comprise more than 80% of all macromolecular speciespresent in the preparation. Preferably, the protein is purified torepresent greater than 90% of all macromolecular species present. Morepreferably the protein is purified to greater than 95%, and mostpreferably the protein is purified to essential homogeneity, whereinother macromolecular species are not detected by conventionaltechniques.

The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of theherpesvirus of the invention in the presence of a heterogeneouspopulation of proteins and other biologics including viruses other thanthe herpesvirus. Thus, under designated immunoassay conditions, thespecified antibodies bind to the herpesvirus antigens and do not bind ina significant amount to other antigens present in the sample. Specificbinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. Forexample, antibodies raised to the human herpesvirus immunogen describedherein can be selected to obtain antibodies specifically immunoreactivewith the herpesvirus proteins and not with other proteins. Theseantibodies recognize proteins homologous to the human herpesvirusprotein. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane [32] for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

“Biological sample” as used herein refers to any sample obtained from aliving organism or from an organism that has died. Examples ofbiological samples include body fluids and tissue specimens.

I. Kaposis's Sarcoma (KS)— Associated Herpesvirus.

This invention provides an isolated DNA molecule which is at least 30nucleotides in length and which uniquely defines a herpesvirusassociated with Kaposi's sarcoma.

In one embodiment the isolated DNA molecule comprises at least a portionof the nucleic acid sequence as shown in FIG. 3A (SEQ ID NO: 1). Inanother embodiment the isolated DNA molecule is a 330 base pair (bp)sequence. In another embodiment the isolated DNA molecule is a 12-50 bpsequence. In another embodiment the isolated DNA molecule is a 30-37 bpsequence.

In another embodiment the isolated DNA molecule is genomic DNA. Inanother embodiment the isolated DNA molecule is cDNA. In anotherembodiment a RNA is derived form the isolated nucleic acid molecule oris capable of hybridizing with the isolated DNA molecule. As used herein“genomic” means both coding and non-coding regions of the isolatednucleic acid molecule.

Further, the DNA molecule above may be associated withlymphoproliferative diseases including, but not limited to: Hodgkin'sdisease, non-Hodgkin's lymphoma, lymphatic leukemia, lymphosarcoma,splenomegaly, reticular cell sarcoma, Sezary's syndrome, mycosisfungoides, central nervous system lymphoma, AIDS related central nervoussystem lymphoma, post-transplant lymphoproliferative disorders, andBurkitt's lymphoma. A lymphoproliferative disorder is characterized asbeing the uncontrolled clonal or polyclonal expansion of lymphocytesinvolving lymph nodes, lymphoid tissue and other organs.

This invention provides an isolated nucleic acid molecule encoding anORF20 (SEQ ID NOs: 22 and 23), ORF21 (SEQ ID NOs:14 and 15), ORF22 (SEQID NOs:16 and 17), ORF23 (SEQ ID NOs:18 and 19), ORF24 (SEQ ID NOs: 20and 21), ORF25 (SEQ ID NOs: 2 and 3), ORF26 (SEQ ID NOs:24 and 25),ORF27 (SEQ ID NOs:26 and 27), ORF28 (SEQ ID NOs:28 and 29), ORF29A (SEQID NOs:30 and 31), ORF29B (SEQ ID NOs:4 and 5), ORF30 (SEQ ID NOs:6 and7), ORF31 (SEQ ID NOs:8 and 9), ORF32 (SEQ ID NOs:32 and 33), ORF33 (SEQID NOs: 10 and 11), ORF34 (SEQ ID NOs: 34 and 35), or ORF35 (SEQ IDNOs:12 AND 13).

This invention provides an isolated polypeptide encoded by ORF20 (SEQ IDNOs: 22 and 23), ORF21 (SEQ ID NOs:14 and 15), ORF22 (SEQ ID NOs:16 and17), ORF23 (SEQ ID NOs:18 and 19), ORF24 (SEQ ID NOs: 20 and 21), ORF25(SEQ ID NOs: 2 and 3), ORF26 (SEQ ID NOs:24 and 25), ORF27 (SEQ IDNOs:26 and 27), ORF28 (SEQ ID NOs:28 and 29), ORF29A (SEQ ID NOs:30 and31), ORF29B (SEQ ID NOs:4 and 5), ORF30 (SEQ ID NOs:6 and 7), ORF31 (SEQID NOs:8 and 9), ORF32 (SEQ ID NOs:32 and 33), ORF33 (SEQ ID NOs: 10 and11), ORF34 (SEQ ID NOs: 34 and 35), or ORF35 (SEQ ID NOs:12 AND 13).

For Example, TK is encoded by ORF 21; glycoprotein H (gH) by ORF 22;major capsid protein (MCP) by ORF 25; virion polypeptide (VP23) by ORF26; and minor capsid protein by ORF 27.

This invention provides for a replicable vector comprising the isolatedDNA molecule of the DNA virus. The vector includes, but is not limitedto: a plasmid, cosmid, λ phage or yeast artificial chromosome (YAC)which contains at least a portion of the isolated nucleic acid molecule.

As an example to obtain these vectors, insert and vector DNA can both beexposed to a restriction enzyme to create complementary ends on bothmolecules which base pair with each other and are then ligated togetherwith DNA ligase. Alternatively, linkers can be ligated to the insert DNAwhich correspond to a restriction site in the vector DNA, which is thendigested with the restriction enzyme which cuts at that site. Othermeans are also available and known to an ordinary skilled practitioner.

Regulatory elements required for expression include promoter or enhancersequences to bind RNA polymerase and transcription initiation sequencesfor ribosome binding. For example, a bacterial expression vectorincludes a promoter such as the lac promoter and for transcriptioninitiation the Shine-Dalgarno sequence and the start codon AUG.Similarly, a eukaryotic expression vector includes a heterologous orhomologous promoter for RNA polymerase II, a downstream polyadenylationsignal, the start codon AUG, and a termination codon for detachment ofthe ribosome. Such vectors may be obtained commercially or assembledfrom the sequences described by methods well-known in the art, forexample the methods described above for constructing vectors in general.

This invention provides a host cell containing the above vector. Thehost cell may contain the isolated DNA molecule artificially introducedinto the host cell. The host cell may be a eukaryotic or bacterial cell(such as E. coli), yeast cells, fungal cells, insect cells and animalcells. Suitable animal cells include, but are not limited to Vero cells,HeLa cells, Cos cells, CV1 cells and various primary mammalian cells.

This invention provides an isolated herpesvirus associated with Kaposi'ssarcoma. In one embodiment the herpesvirus comprises at least a portionof a nucleotide sequence as shown in FIGS. 3A (SEQ ID NO: 1).

In one embodiment the herpesvirus may be a DNA virus. In anotherembodiment the herpesvirus may be a Herpesviridae. In another embodimentthe herpesvirus may be a gammaherpesvirinae. The classification of theherpesvirus may vary based on the phenotypic or molecularcharacteristics which are known to those skilled in the art.

This invention provides an isolated DNA virus wherein the viral DNA isabout 270 kb in size, wherein the viral DNA encodes a thymidine kinase,and wherein the viral DNA is capable of selectively hybridizing to anucleic acid probe selected from the group consisting of SEQ ID NOs:38-40.

The KS-associated human herpesvirus of the invention is associated withKS and is involved in the etiology of the disease. The taxonomicclassification of the virus has not yet been made and will be based onphenotypic or molecular characteristics known to those of skill in theart. However, the novel KS-associated virus is a DNA virus that appearsto be related to the Herpesviridae family and the gammaherpesvirinaesubfamily, on the basis of nucleic acid homology.

A. Sequence Identity of the Viral DNA and its Proteins.

The human herpesvirus of the invention is not limited to the virushaving the specific DNA sequences described herein. The KS-associatedhuman herpesvirus DNA shows substantial sequence identity, as definedabove, to the viral DNA sequences described herein. DNA from the humanherpesvirus typically selectively hybridizes to one or more of thefollowing three nucleic acid probes:

Probe 1 (SEQ ID NO: 38) AGCCGAAAGG ATTCCACCAT TGTGCTCGAA TCCAACGGATTTGACCCCGT GTTCCCCATG GTCGTGCCGC AGCAACTGGGGCACGCTATT CTGCAGCAGC TGTTGGTGTA CCACATCTACTCCAAAATAT CGGCCGGGGC CCCGGATGAT GTAAATATGGCGGAACTTGA TCTATATACC ACCAATGTGT CATTTATGGGGCGCACATAT CGTCTGGACG TAGACAACAC GGA Probe 2 (SEQ ID NO: 39):GAAATTACCC ACGAGATCGC TTCCCTGCAC ACCGCACTTGGCTACTCATC AGTCATCGCC CCGGCCCACG TGGCCGCCATAACTACAGAC ATGGGAGTAC ATTGTCAGGA CCTCTTTATGATTTTCCCAG GGGACGCGTA TCAGGACCGC CAGCTGCATGACTATATCAA AATGAAAGCG GGCGTGCAAA CCGGCTCACCGGGAAACAGA ATGGATCACG TGGGATACAC TGCTGGGGTTCCTCGCTGCG AGAACCTGCC CGGTTTGAGT CATGGTCAGCTGGCAACCTG CGAGATAATT CCCACGCCGG TCACATCTGA CGTTGCCTProbe 3 (SEQ ID NO: 40): AACACGTCAT GTGCAGGAGT GACATTGTGC CGCGGAGAAACTCAGACCGC ATCCCGTAAC CACACTGAGT GGGAAAATCTGCTGGCTATG TTTTCTGTGA TTATCTATGC CTTAGATCAC AACTGTCACC CG

Hybridization of a viral DNA to the nucleic acid probes listed above isdetermined by using standard nucleic acid hybridization techniques asdescribed herein. In particular, PCR amplification of a viral genome canbe carried out using the following three sets of PCR primers:

1) AGCCGAAAGGATTCCACCAT; (SEQ ID NO: 41) TCCGTGTTGTCTACGTCCAG(SEQ ID NO: 48) 2) GAAATTACCCACGAGATCGC; (SEQ ID NO: 42)AGGCAACGTCAGATGTGA (SEQ ID NO: 49) 3) AACACGTCATGTGCAGGAGTGAC;(SEQ ID NO: 43) CGGGTGACAGTTGTGATCTAAGG (SEQ ID NO: 50)

In PCR techniques, oligonucleotide primers, as listed above,complementary to the two 3′ borders of the DNA region to be amplifiedare synthesized. The polymerase chain reaction is then carried out usingthe two primers. See PCR Protocols: A Guide to Methods and Applications[74]. Following PCR amplification, the PCR-amplified regions of a viralDNA can be tested for their ability to hybridize to the three specificnucleic acid probes listed above. Alternatively, hybridization of aviral DNA to the above nucleic acid probes can be performed by aSouthern blot procedure without viral DNA amplification and understringent hybridization conditions as described herein.

Oligonucleotides for use as probes or PCR primers are chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage and Carruthers [19] using an automatedsynthesizer, as described in Needham-VanDevanter [69]. Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson, J. D. and Regnier, F. E.[75A]. The sequence of the synthetic oligonucleotide can be verifiedusing the chemical degradation method of Maxam, A. M. and Gilbert, W.[63].

B. Isolation and Propagation of KS-Inducing Strains of the HumanHerpesvirus

Using conventional methods, the human herpesvirus can be propagated invitro. For example, standard techniques for growing herpes viruses aredescribed in Ablashi, D. V. [1]. Briefly, PHA stimulated cord bloodmononuclear cells, macrophage, neuronal, or glial cell lines arecocultivated with cerebrospinal fluid, plasma, peripheral bloodleukocytes, or tissue extracts containing viral infected cells orpurified virus. The recipient cells are treated with 5 μg/ml polybrenefor 2 hours at 37° C. prior to infection.

Infected cells are observed by demonstrating morphological changes, aswell as being positive for antigens from the human herpesvirus by usingmonoclonal antibodies immunoreactive with the human herpes virus in animmunofluorescence assay.

For virus isolation, the virus is either harvested directly from theculture fluid by direct centrifugation, or the infected cells areharvested, homogenized or lysed and the virus is separated from cellulardebris and purified by standard methods of isopycnic sucrose densitygradient centrifugation.

One skilled in the art may isolate and propagate the DNA herpesvirusassociated with Kaposi's sarcoma (KSHV) employing the followingprotocol. Long-term establishment of a B lymphoid cell line infectedwith the KSHV from body-cavity based lymphomas (RCC-1 or HBL-6) isprepared extracting DNA from the Lymphoma tissue using standardtechniques [27, 49, 66].

The KS associated herpesvirus may be isolated from the cell DNA in thefollowing manner. An infected cell line (HBL-6 RCC-1), which can belysed using standard methods such as hyposomatic shocking and Douncehomogenization, is first pelleted at 2000×g for 10 minutes, thesupernatant is removed and centrifuged again at 10,000×g for 15 minutesto remove nuclei and organelles. The supernatant is filtered through a0.45μ filter and centrifuged again at 100,000×g for 1 hour to pellet thevirus. The virus can then be washed and centrifuged again at 100,000×gfor 1 hour.

The DNA is tested for the presence of the KSHV by Southern blotting andPCR using the specific probes as described hereinafter. Fresh lymphomatissue containing viable infected cells is simultaneously filtered toform a single cell suspension by standard techniques [49, 66]. The cellsare separated by standard Ficoll-Plaque centrifugation and lymphocytelayer is removed. The lymphocytes are then placed at >1×10⁶ cells/mlinto standard lymphocyte tissue culture medium, such as RMP 1640supplemented with 10% fetal calf serum. Immortalized lymphocytescontaining the KSHV virus are indefinitely grown in the culture mediawhile nonimmortilized cells die during course of prolonged cultivation.

Further, the virus may be propagated in a new cell line by removingmedia supernatant containing the virus from a continuously infected cellline at a concentration of >1×10⁶ cells/ml. The media is centrifuged at2000×g for 10 minutes and filtered through a 0.45μ filter to removecells. The media is applied in a 1:1 volume with cells growing at >1×10⁶cells/ml for 48 hours. The cells are washed and pelleted and placed infresh culture medium, and tested after 14 days of growth.

RCC-1 and RCC-1_(2F5) were deposited on Oct. 19, 1994 under ATCCAccession No. CRL 11734 and CRL 11735, respectively, pursuant to theBudapest Treaty on the International Deposit of Microorganisms for thePurposes of Patent Procedure with the Patent Culture Depository of theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 U.S.A.

HBL-6 was deposited on Nov. 18, 1994 under ATCC Accession No. CRL 11762pursuant to the Budapest Treaty on the International Deposit ofMicroorganisms for the Purposes of Patent Procedure with the PatentCulture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A.

C. Immunological Identity of the Virus

The KS-associated human herpesvirus can also be describedimmunologically. KS-associated human herpesviruses are selectivelyimmunoreactive to antisera generated against a defined immunogen such asthe viral major capsid protein depicted in Seq. ID No. 12, herein.Immunoreactivity is determined in an immunoassay using a polyclonalantiserum which was raised to the protein which is encoded by the aminoacid sequence or nucleic acid sequence of SEQ ID NOs: 18-20. Thisantiserum is selected to have low crossreactivity against other herpesviruses and any such crossreactivity is removed by immunoabsorptionprior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the proteinwhich is encoded by the amino acid sequence or nucleic acid of SEQ IDNOs: 18-20 is isolated as described herein. For example, recombinantprotein can be produced in a mammalian cell line. An inbred strain ofmice such as balb/c is immunized with the protein which is encoded bythe amino acid sequence or nucleic acid of SEQ ID NOs: 2-37 using astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see [32], supra). Alternatively, a syntheticpeptide derived from the sequences disclosed herein and conjugated to acarrier protein can be used an immunogen. Polyclonal sera are collectedand titered against the immunogen protein in an immunoassay, forexample, a solid phase immunoassay with the immunogen immobilized on asolid support. Polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against other viruses ofthe gammaherpesvirinae subfamily, particularly human herpes virus types1-7, by using a standard immunoassay as described in [32], supra. Theseother gammaherpesvirinae virus can be isolated by standard techniquesfor isolation herpes viruses as described herein.

The ability of the above viruses to compete with the binding of theantisera to the immunogen protein is determined. The percentcrossreactivity for other viruses is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the other viruses listed above is selected and pooled. Thecross-reacting antibodies are then removed from the pooled antisera byimmunoabsorption with the above-listed viruses.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay procedure as described above to compare an unknownvirus preparation to the specific KS herpesvirus preparation describedherein and containing the nucleic acid sequence described in SEQ ID NOs:2-37. In order to make this comparison, the immunogen protein which isencoded by the amino acid sequence or nucleic acid of SEQ ID NOs: 2-37is the labeled antigen and the virus preparations are each assayed at awide range of concentrations. The amount of each virus preparationrequired to inhibit 50% of the binding of the antisera to the labeledimmunogen protein is determined. Those viruses that specifically bind toan antibody generated to an immunogen consisting of the protein of SEQID NOs: 2-37 are those virus where the amount of virus needed to inhibit50% of the binding to the protein does not exceed an established amount.This amount is no more than 10 times the amount of the virus that isneeded for 50% inhibition for the KS-associated herpesvirus containingthe DNA sequence of SEQ ID NO: 1. Thus, the KS-associated herpesvirusesof the invention can be defined by immunological comparison to thespecific strain of the KS-associated herpesvirus for which nucleic acidsequences are provided herein.

This invention provides, a nucleic acid molecule of at least 14nucleotides capable of specifically-hybridizing with the isolated DNAmolecule. In one embodiment, the molecule is DNA. In another embodiment,the molecule is RNA. In another embodiment the nucleic acid molecule maybe 14-20 nucleotides in length. In another embodiment the nucleic acidmolecule may be 16 nucleotides in length.

This invention provides, a nucleic acid molecule of at least 14nucleotides capable of specifically hybridizing with a nucleic acidmolecule which is complementary to the isolated DNA molecule. In oneembodiment, the molecule is DNA. In another embodiment, the molecule isRNA.

The nucleic acid molecule of at least 14 nucleotides may hybridize withmoderate stringency to at least a portion of a nucleic acid moleculewith a sequence shown in FIGS. 3A-3F (SEQ ID NOs: 1, and 36-40).

High stringent hybridization conditions are selected at about 5° C.lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa perfectly matched probe. Typically, stringent conditions will be thosein which the salt concentration is at least about 0.02 molar at pH 7 andthe temperature is at least about 60° C. As other factors maysignificantly affect the stringency of hybridization, including, amongothers, base composition and size of the complementary strands, thepresence of organic solvents, ie. salt or formamide concentration, andthe extent of base mismatching, the combination of parameters is moreimportant than the absolute measure of any one. For Example highstringency may be attained for example by overnight hybridization atabout 68° C. in a 6×SSC solution, washing at room temperature with 6×SSCsolution, followed by washing at about 68° C. in a 6×SSC in a 0.6×SSXsolution.

Hybridization with moderate stringency may be attained for exampleby: 1) filter pre-hybridizing and hybridizing with a solution of 3×sodium chloride, sodium citrate (SSC), 50% formamide, 0.1M Tris bufferat Ph 7.5, 5×Denhardt's solution; 2.) pre-hybridization at 37° C. for 4hours; 3) hybridization at 37° C. with amount of labelled probe equal to3,000,000 cpm total for 16 hours; 4) wash in 2×SSC and 0.1% SDSsolution; 5) wash 4× for 1 minute each at room temperature at 4× at 60°C. for 30 minutes each; and 6) dry and expose to film.

The phrase “selectively hybridizing to” refers to a nucleic acid probethat hybridizes, duplexes or binds only to a particular target DNA orRNA sequence when the target sequences are present in a preparation oftotal cellular DNA or RNA. By selectively hybridizing it is meant that aprobe binds to a given target in a manner that is detectable in adifferent manner from non-target sequence under high stringencyconditions of hybridization, in a different “Complementary” or “target”nucleic acid sequences refer to those nucleic acid sequences whichselectively hybridize to a nucleic acid probe. Proper annealingconditions depend, for example, upon a probe's length, base composition,and the number of mismatches and their position on the probe, and mustoften be determined empirically. For discussions of nucleic acid probedesign and annealing conditions, see, for example, Sambrook at al., [81]or Ausubel, F., et al., [8].

It will be readily understood by those skilled in the art and it isintended here, that when reference is made to particular sequencelistings, such reference includes sequences which substantiallycorrespond to its complementary sequence and those described includingallowances for minor sequencing errors, single base changes, deletions,substitutions and the like, such that any such sequence variationcorresponds to the nucleic acid sequence of the pathogenic organism ordisease marker to which the relevant sequence listing relates.

Nucleic acid probe technology is well known to those skilled in the artwho readily appreciate that such probes may vary greatly in length andmay be labeled with a detectable label, such as a radioisotope orfluorescent dye, to facilitate detection of the probe. DNA probemolecules may be produced by insertion of a DNA molecule having thefull-length or a fragment of the isolated nucleic acid molecule of theDNA virus into suitable vectors, such as plasmids or bacteriophages,followed by transforming into suitable bacterial host cells, replicationin the transformed bacterial host cells and harvesting of the DNAprobes, using methods well known in the art. Alternatively, probes maybe generated chemically from DNA synthesizers.

DNA virus nucleic acid rearrangements/mutations may be detected bySouthern blotting, single stranded conformational polymorphism gelelectrophoresis (SSCP), PCR or other DNA based techniques, or for RNAspecies by Northern blotting, PCR or other RNA-based techniques.

RNA probes may be generated by inserting the full length or a fragmentof the isolated nucleic acid molecule of the DNA virus downstream of abacteriophage promoter such as T3, T7 or SP6. Large amounts of RNA probemay be produced by incubating the labeled nucleotides with a linearizedisolated nucleic acid molecule of the DNA virus or its fragment where itcontains an upstream promoter in the presence of the appropriate RNApolymerase.

As defined herein nucleic acid probes may be DNA or RNA fragments. DNAfragments can be prepared, for example, by digesting plasmid DNA, or byuse of PCR, or synthesized by either the phosphoramidite methoddescribed by Beaucage and Carruthers, [19], or by the triester methodaccording to Matteucci, et al., [62], both incorporated herein byreference. A double stranded fragment may then be obtained, if desired,by annealing the chemically synthesized single strands together underappropriate conditions or by synthesizing the complementary strand usingDNA polymerase with an appropriate primer sequence. Where a specificsequence for a nucleic acid probe is given, it is understood that thecomplementary strand is also identified and included. The complementarystrand will work equally well in situations where the target is adouble-stranded nucleic acid. It is also understood that when a specificsequence is identified for use a nucleic probe, a subsequence of thelisted sequence which is 25 basepairs or more in length is alsoencompassed for use as a probe.

The DNA molecules of the subject invention also include DNA moleculescoding for polypeptide analogs, fragments or derivatives of antigenicpolypeptides which differ from naturally-occurring forms in terms of theidentity or location of one or more amino acid residues (deletionanalogs containing less than all of the residues specified for theprotein, substitution analogs wherein one or more residues specified arereplaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors.

This invention provides for an isolated DNA molecule which encodes atleast a portion of a Kaposi's sarcoma associated herpesvirus: virionpolypeptide 23, major capsid protein, capsid proteins, thymidine kinase,or tegument protein.

This invention also provides a method of producing a polypeptide encodedby isolated DNA molecule, which comprises growing the above host vectorsystem under suitable conditions permitting production of thepolypeptide and recovering the polypeptide so produced.

This invention provides an isolated peptide encoded by the isolated DNAmolecule associated with Kaposi's sarcoma. In one embodiment the peptidemay be a polypeptide. Further, this invention provides a host cell whichexpresses the polypeptide of isolated DNA molecule.

In one embodiment the isolated peptide or polypeptide is encoded by atleast a portion of an isolated DNA molecule. In another embodiment theisolated peptide or polypeptide is encoded by at least a portion of anucleic acid molecule with a sequence as set forth in (SEQ ID NOs:2-37).

Further, the isolated peptide or polypeptide encoded by the isolated DNAmolecule may be linked to a second nucleic acid molecule to form afusion protein by expression in a suitable host cell. In one embodimentthe second nucleic acid molecule encodes beta-galactosidase. Othernucleic acid molecules which are used to form a fusion protein are knownto those skilled in the art.

This invention provides an antibody which specifically binds to thepeptide or polypeptide encoded by the isolated DNA molecule. In oneembodiment the antibody is a monoclonal antibody. In another embodimentthe antibody is a polyclonal antibody.

The antibody or DNA molecule may be labelled with a detectable markerincluding, but not limited to: a radioactive label, or a colorimetric, aluminescent, or a fluorescent marker, or gold. Radioactive labelsinclude, but are not limited to: ³H, ¹⁴C, ³²P, ³³P; ³⁵S, ³⁶Cl, ⁵¹Cr,⁵⁷Co, ⁵⁹Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Fluorescent markersinclude but are not limited to: fluorescein, rhodamine and auramine.Colorimetric markers include, but are not limited to: biotin, anddigoxigenin. Methods of producing the polyclonal or monoclonal antibodyare known to those of ordinary skill in the art.

Further, the antibody or nucleic acid molecule complex may be detectedby a second antibody which may be linked to an enzyme, such as alkalinephosphatase or horseradish peroxidase. Other enzymes which may beemployed are well known to one of ordinary skill in the art.

This invention provides a method to select specific regions on thepolypeptide encoded by the isolated DNA molecule of the DNA virus togenerate antibodies. The protein sequence may be determined from thecDNA sequence. Amino acid sequences may be analyzed by methods wellknown to those skilled in the art to determine whether they producehydrophobic or hydrophilic regions in the proteins which they build. Inthe case of cell membrane proteins, hydrophobic regions are well knownto form the part of the protein that is inserted into the lipid bilayerof the cell membrane, while hydrophilic regions are located on the cellsurface, in an aqueous environment. Usually, the hydrophilic regionswill be more immunogenic than the hydrophobic regions. Therefore thehydrophilic amino acid sequences may be selected and used to generateantibodies specific to polypeptide encoded by the isolated nucleic acidmolecule encoding the DNA virus. The selected peptides may be preparedusing commercially available machines. As an alternative, DNA, such as acDNA or a fragment thereof, may be cloned and expressed and theresulting polypeptide recovered and used as an immunogen.

Polyclonal antibodies against these peptides may be produced byimmunizing animals using the selected peptides. Monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody.Alternatively, monoclonal antibodies may be produced by in vitrotechniques known to a person of ordinary skill in the art. Theseantibodies are useful to detect the expression of polypeptide encoded bythe isolated DNA molecule of the DNA virus in living animals, in humans,or in biological tissues or fluids isolated from animals or humans.

II. Immunoassays

The antibodies raised against the viral strain or peptides may bedetectably labelled, utilizing conventional labelling techniqueswell-known to the art. Thus, the antibodies may be radiolabelled using,for example, radioactive isotopes such as ³H, ¹²⁵I, ¹³¹I, and ³⁵S.

The antibodies may also be labelled using fluorescent labels, enzymelabels, free radical labels, or bacteriophage labels, using techniquesknown in the art. Typical fluorescent labels include fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, alophycocyanin,and Texas Red.

Since specific enzymes may be coupled to other molecules by covalentlinks, the possibility also exists that they might be used as labels forthe production of tracer materials. Suitable enzymes include alkalinephosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase,maleate dehydrogenase, and peroxidase. Two principal types of enzymeimmunoassay are the enzyme-linked immunosorbent assay (ELISA), and thehomogeneous enzyme immunoassay, also known as enzyme-multipliedimmunoassay (EMIT, Syva Corporation, Palo Alto, Calif.). In the ELISAsystem, separation may be achieved, for example, by the use ofantibodies coupled to a solid phase. The EMIT system depends ondeactivation of the enzyme in the tracer-antibody complex; the activitycan thus be measured without the need for a separation step.

Additionally, chemiluminescent compounds may be used as labels. Typicalchemiluminescent compounds include luminol, isoluminol, aromaticacridinium esters, imidazoles, acridinium salts, and oxalate esters.Similarly, bioluminescent compounds may be utilized for labelling, thebioluminescent compounds including luciferin, luciferase, and aequorin.

Once labeled, the antibody may be employed to identify and quantifyimmunologic counterparts (antibody or antigenic polypeptide) utilizingtechniques well-known to the art.

A description of a radioimmunoassay (RIA) may be found in LaboratoryTechniques in Biochemistry and Molecular Biology [52], with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard, T., incorporated by reference herein.

A description of general immunometric assays of various types can befound in the following U.S. Pat. Nos. 4,376,110 (David et al.) or4,098,876 (Piasio).

A. Assays for Viral Antigens

In addition to the detection of the causal agent using nucleic acidhybridization technology, one can use immunoassays to detect for thevirus, specific peptides, or for antibodies to the virus or peptides. Ageneral overview of the applicable technology is in Harlow and Lane[32], incorporated by reference herein.

In one embodiment, antibodies to the human herpesvirus can be used todetect the agent in the sample. In brief, to produce antibodies to theagent or peptides, the sequence being targeted is expressed intransfected cells, preferably bacterial cells, and purified. The productis injected into a mammal capable of producing antibodies. Eithermonoclonal or polyclonal antibodies (as well as any recombinantantibodies) specific for the gene product can be used in variousimmunoassays. Such assays include competitive immunoassays,radioimmunoassays, Western blots, ELISA, indirect immunofluorescentassays and the like. For competitive immunoassays, see Harlow and Lane[32] at pages 567-573 and 584-589.

Monoclonal antibodies or recombinant antibodies may be obtained byvarious techniques familiar to those skilled in the art. Briefly, spleencells or other lymphocytes from an animal immunized with a desiredantigen are immortalized, commonly by fusion with a myeloma cell (see,Kohler and Milstein [50], incorporated herein by reference). Alternativemethods of immortalization include transformation with Epstein BarrVirus, oncogenes, or retroviruses, or other methods well known in theart. Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monoclonal antibodies produced by such cellsmay be enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. New techniques using recombinantphage antibody expression systems can also be used to generatemonoclonal antibodies. See for example: McCafferty, J et al. [64];Hoogenboom, H. R. et al. [39]; and Marks, J. D. et al., [60].

Such peptides may be produced by expressing the specific sequence in arecombinantly engineered cell such as bacteria, yeast, filamentousfungal, insect (especially employing baculoviral vectors), and mammaliancells. Those of skill in the art are knowledgeable in the numerousexpression systems available for expression of herpes virus protein.

Briefly, the expression of natural or synthetic nucleic acids encodingviral protein will typically be achieved by operably linking the desiredsequence or portion thereof to a promoter (which is either constitutiveor inducible), and incorporated into an expression vector. The vectorsare suitable for replication or integration in either prokaryotes oreukaryotes. Typical cloning vectors contain antibiotic resistancemarkers, genes for selection of transformants, inducible or regulatablepromoter regions, and translation terminators that are useful for theexpression of viral genes.

Methods for the expression of cloned genes in bacteria are also wellknown. In general, to obtain high level expression of a cloned gene in aprokaryotic system, it is advisable to construct expression vectorscontaining a strong promoter to direct mRNA transcription. The inclusionof selection markers in DNA vectors transformed in E. coli is alsouseful. Examples of such markers include genes specifying resistance toantibiotics. See [81] supra, for details concerning selection markersand promoters for use in E. coli. Suitable eukaryote hosts may includeplant cells, insect cells, mammalian cells, yeast, and filamentousfungi.

Methods for characterizing naturally processed peptides bound to MHC(major histocompatibility complex) I molecules have been developed. See,Falk et al. [24], and PCT publication No. WO 92/21033 published Nov. 26,1992, both of which are incorporated by reference herein. Typically,these methods involve isolation of MHC class I molecules byimmunoprecipitation or affinity chromatography from an appropriate cellor cell line. Other methods involve direct amino acid sequencing of themore abundant peptides in various HPLC fractions by known automaticsequencing of peptides eluted from Class I molecules of the B cell type(Jardetzkey, et al. [45], incorporated by reference herein, and of thehuman MHC class I molecule, HLA-A2.1 type by mass spectrometry (Hunt, etal. [40], incorporated by reference herein). See also, Rötzschke andFalk [79], incorporated by reference herein for a general review of thecharacterization of naturally processed peptides in MHC class I.Further, Marloes, et al. [61], incorporated by reference herein,describe how class I binding motifs can be applied to the identificationof potential viral immunogenic peptides in vitro.

The peptides described herein produced by recombinant technology may bepurified by standard techniques well known to those of skill in the art.Recombinantly produced viral sequences can be directly expressed orexpressed as a fusion protein. The protein is then purified by acombination of cell lysis (e.g., sonication) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredpeptide.

The proteins may be purified to substantial purity by standardtechniques well known in the art, including selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, Scopes, R.[84], incorporated herein by reference.

B. Serological Tests for the Presence of Antibodies to the HumanHerpesvirus.

This invention further embraces diagnostic kits for detecting thepresence of a KS agent in biological samples, such as serum or solidtissue samples, comprising a container containing antibodies to thehuman herpesvirus, and instructional material for performing the test.Alternatively, inactivated viral particles or peptides or viral proteinsderived from the human herpesvirus may be used in a diagnostic kit todetect for antibodies specific to the KS associated human herpesvirus.

Diagnostic kits for detecting the presence of a KS agent in tissuesamples, such as skin samples or samples of other affected tissue,comprising a container containing a nucleic acid sequence specific forthe human herpesvirus and instructional material for detecting theKS-associated herpesvirus are also included. A container containingnucleic acid primers to any one of such sequences is optionally includedas are antibodies to the human herpesvirus as described herein.

Antibodies reactive with antigens of the human herpesvirus can also bemeasured by a variety of immunoassay methods that are similar to theprocedures described above for measurement of antigens. For a review ofimmunological and immunoassay procedures applicable to the measurementof antibodies by immunoassay techniques, see Basic and ClinicalImmunology 7th Edition [12], and [32], supra.

In brief, immunoassays to measure antibodies reactive with antigens ofthe KS-associated human herpesvirus can be either competitive ornoncompetitive binding assays. In competitive binding assays, the sampleanalyte competes with a labeled analyte for specific binding sites on acapture agent bound to a solid surface. Preferably the capture agent isa purified recombinant human herpesvirus protein produced as describedabove. Other sources of human herpesvirus proteins, including isolatedor partially purified naturally occurring protein, may also be used.Noncompetitive assays are typically sandwich assays, in which the sampleanalyte is bound between two analyte-specific binding reagents. One ofthe binding agents is used as a capture agent and is bound to a solidsurface. The second binding agent is labelled and is used to measure ordetect the resultant complex by visual or instrument means. A number ofcombinations of capture agent and labelled binding agent can be used. Avariety of different immunoassay formats, separation techniques andlabels can be also be used similar to those described above for themeasurement of the human herpesvirus antigens.

Hemagglutination Inhibition (HI) and Complement Fixation (CF) which aretwo laboratory tests that can be used to detect infection with humanherpesvirus by testing for the presence of antibodies against the virusor antigens of the virus.

Serological methods can be also be useful when one wishes to detectantibody to a specific variant. For example, one may wish to see howwell a vaccine recipient has responded to the new variant.

Alternatively, one may take serum from a patient to see which variantthe patient responds to the best.

This invention provides an antagonist capable of blocking the expressionof the peptide or polypeptide encoded by the isolated DNA molecule. Inone embodiment the antagonist is capable of hybridizing with a doublestranded DNA molecule. In another embodiment the antagonist is a triplexoligonucleotide capable of hybridizing to the DNA molecule. In anotherembodiment the triplex oligonucleotide is capable of binding to at leasta portion of the isolated DNA molecule with a nucleotide sequence asshown in FIG. 3A-3F (SEQ ID NOs: 1, and 36-40).

This invention provides an antisense molecule capable of hybridizing tothe isolated DNA molecule. In one embodiment the antisense molecule isDNA. In another embodiment the antisense molecule is RNA.

The antisense molecule may be DNA or RNA or variants thereof (i.e. DNAor RNA with a protein backbone). The present invention extends to thepreparation of antisense nucleotides and ribozymes that may be used tointerfere with the expression of the receptor recognition proteins atthe translation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. In the cell, theyhybridize to that mRNA, forming a double stranded molecule. The celldoes not translate an mRNA in this double-stranded 35 form. Therefore,antisense nucleic acids interfere with the expression of mRNA intoprotein. Oligomers of about fifteen nucleotides and molecules thathybridize to the AUG initiation codon are particularly efficient, sincethey are easy to synthesize and are likely to pose fewer problems thanlarger molecules upon introduction to cells.

This invention provides a transgenic nonhuman mammal which comprises atleast a portion of the isolated DNA molecule introduced into the mammalat an embryonic stage. Methods of producing a transgenic nonhuman mammalare known to those skilled in the art.

This invention provides a cell line containing the isolated KSassociated herpesvirus of the subject invention. In one embodiment theisolated DNA molecule is artificially introduced into the cell. Celllines include, but are not limited to: fibroblasts, such as HFF,NIH/3T3; Epithelial cells, such as 5637; lymphocytes, such as FCB;T-cells, such as CCRF-CEM (ATCC CCL 119); B-cells, such as BJAB and Raji(ATCC CCL 86); and myeloid cells such as K562 (ATCC CCL 243); Vero cellsand carcinoma cells. Methods of producing such cell lines are known tothose skilled in the art. In one embodiment the isolated KS associatedherpesvirus is introduced into a RCC-1 cell line.

III. In Vitro Diagnostic Assays for the Detection of KS

This invention provides a method of diagnosing Kaposi's sarcoma in asubject which comprises: (a) obtaining a nucleic acid molecule from atumor lesion of the subject: (b) contacting the nucleic acid moleculewith a labelled nucleic acid molecule of at least 15 nucleotides capableof specifically hybridizing with the isolated DNA, under hybridizingconditions; and (c) determining the presence of the nucleic acidmolecule hybridized, the presence of which is indicative of Kaposi'ssarcoma in the subject, thereby diagnosing Kaposi's sarcoma in thesubject.

In one embodiment the DNA molecule from the tumor lesion is amplifiedbefore step (b). In another embodiment PCR is employed to amplify thenucleic acid molecule. Methods of amplifying nucleic acid molecules areknown to those skilled in the art.

A person of ordinary skill in the art will be able to obtain appropriateDNA sample for diagnosing Kaposi's sarcoma in the subject. The DNAsample obtained by the above described method may be cleaved byrestriction enzyme. The uses of restriction enzymes to cleave DNA andthe conditions to perform such cleavage are well-known in the art.

In the above described methods, a size fractionation may be employedwhich is effected by a polyacrylamide gel. In one embodiment, the sizefractionation is effected by an agarose gel. Further, transferring theDNA fragments into a solid matrix may be employed before a hybridizationstep. One example of such solid matrix is nitrocellulose paper.

This invention provides a method of diagnosing Kaposi's sarcoma in asubject which comprises: (a) obtaining a nucleic acid molecule from asuitable bodily fluid of the subject; (b) contacting the nucleic acidmolecule with a labelled nucleic acid molecules of at least 15nucleotides capable of specifically hybridizing with the isolated DNA,under hybridizing conditions; and (c) determining the presence of thenucleic acid molecule hybridized, the presence of which is indicative ofKaposi's sarcoma in the subject, thereby diagnosing Kaposi's sarcoma inthe subject.

This invention provides a method of diagnosing a DNA virus in a subject,which comprises (a) obtaining a suitable bodily fluid sample from thesubject, (b) contacting the suitable bodily fluid of the subject to asupport having already bound thereto a Kaposi's sarcoma antibody, so asto bind the Kaposi's sarcoma antibody to a specific Kaposi's sarcomaantigen, (c) removing unbound bodily fluid from the support, and (d)determining the level of Kaposi's sarcoma antibody bound by the Kaposi'ssarcoma antigen, thereby diagnosing the subject for Kaposi's sarcoma.

This invention provides a method of diagnosing Kaposi's sarcoma in asubject, which comprises (a) obtaining a suitable bodily fluid samplefrom the subject, (b) contacting the suitable bodily fluid of thesubject to a support having already bound thereto a Kaposi's sarcomaantigen, so as to bind Kaposi's sarcoma antigen to a specific Kaposi'ssarcoma antibody, (c) removing unbound bodily fluid from the support,and (d) determining the level of the Kaposi's sarcoma antigen bound bythe Kaposi's sarcoma antibody, thereby diagnosing Kaposi's sarcoma.

This invention provides a method of detecting expression of a DNA virusassociated with Kaposi's sarcoma in a cell which comprises obtainingtotal cDNA obtained from the cell, contacting the cDNA so obtained witha labelled DNA molecule under hybridizing conditions, determining thepresence of cDNA hybridized to the molecule, and thereby detecting theexpression of the DNA virus. In one embodiment mRNA is obtained from thecell to detect expression of the DNA virus.

The suitable bodily fluid sample is any bodily fluid sample which wouldcontain Kaposi's sarcoma antibody, antigen or fragments thereof. Asuitable bodily fluid includes, but is not limited to: serum, plasma,cerebrospinal fluid, lymphocytes, urine, transudates, or exudates. Inthe preferred embodiment, the suitable bodily fluid sample is serum orplasma. In addition, the bodily fluid sample may be cells from bonemarrow, or a supernatant from a cell culture. Methods of obtaining asuitable bodily fluid sample from a subject are known to those skilledin the art. Methods of determining the level of antibody or antigeninclude, but are not limited to: ELISA, IFA, and Western blotting. Othermethods are known to those skilled in the art. Further, a subjectinfected with a DNA virus associated with Kaposi's sarcoma may bediagnosed with the above described methods.

The detection of the human herpesvirus and the detection ofvirus-associated KS are essentially identical processes. The basicprinciple is to detect the virus using specific ligands that bind to thevirus but not to other proteins or nucleic acids in a normal human cellor its environs. The ligands can either be nucleic acid or antibodies.The ligands can be naturally occurring or genetically or physicallymodified such as nucleic acids with non-natural or antibody derivatives,i.e., Fab or chimeric antibodies. Serological tests for detection ofantibodies to the virus may also be performed by using protein antigensobtained from the human herpesvirus, and described herein.

Samples can be taken from patients with KS or from patients at risk forKS, such as AIDS patients. Typically the samples are taken from blood(cells, serum and/or plasma) or from solid tissue samples such as skinlesions. The most accurate diagnosis for KS will occur if elevatedtiters of the virus are detected in the blood or in involved lesions. KSmay also be indicated if antibodies to the virus are detected and ifother diagnostic factors for KS is present.

A. Nucleic Acid Assays.

The diagnostic assays of the invention can be nucleic acid assays suchas nucleic acid hybridization assays and assays which detectamplification of specific nucleic acid to detect for a nucleic acidsequence of the human herpesvirus described herein.

Accepted means for conducting hybridization assays are known and generaloverviews of the technology can be had from a review of Nucleic AcidHybridization: A Practical Approach [72]; Hybridization of Nucleic AcidsImmobilized on Solid Supports [41]; Analytical Biochemistry [4] andInnis et al., PCR Protocols [74], supra, all of which are incorporatedby reference herein.

If PCR is used in conjunction with nucleic acid hybridization, primersare designed to target a specific portion of the nucleic acid of theherpesvirus. For example, the primers set forth in SEQ ID NOs: 38-40 maybe used to target detection of regions of the herpesvirus genomeencoding ORF 25 homologue-ORF 32 homologue. From the informationprovided herein, those of skill in the art will be able to selectappropriate specific primers.

Target specific probes may be used in the nucleic acid hybridizationdiagnostic assays for KS. The probes are specific for or complementaryto the target of interest. For precise allelic differentiations, theprobes should be about 14 nucleotides long and preferably about 20-30nucleotides. For more general detection of the human herpesvirus of theinvention, nucleic acid probes are about 50 to about 1000 nucleotides,most preferably about 200 to about 400 nucleotides.

A sequence is “specific” for a target organism of interest if itincludes a nucleic acid sequence which when detected is determinative ofthe presence of the organism in the presence of a heterogeneouspopulation of proteins and other biologics. A specific nucleic acidprobe is targeted to that portion of the sequence which is determinativeof the organism and will not hybridize to other sequences especiallythose of the host where a pathogen is being detected.

The specific nucleic acid probe can be RNA or DNA polynucleotide oroligonucleotide, or their analogs. The probes may be single or doublestranded nucleotides. The probes of the invention may be synthesizedenzymatically, using methods well known in the art (e.g., nicktranslation, primer extension, reverse transcription, the polymerasechain reaction, and others) or chemically (e.g., by methods such as thephosphoramidite method described by Beaucage and Carruthers [19], or bythe triester method according to Matteucci, et al. [62], bothincorporated herein by reference).

The probe must be of sufficient length to be able to form a stableduplex with its target nucleic acid in the sample, i.e., at least about14 nucleotides, and may be longer (e.g., at least about 50 or 100 basesin length). Often the probe will be more than about 100 bases in length.For example, when probe is prepared by nick-translation of DNA in thepresence of labeled nucleotides the average probe length may be about100-600 bases.

As noted above, the probe will be capable of specific hybridization to aspecific KS-associated herpes virus nucleic acid. Such “specifichybridization” occurs when a probe hybridizes to a target nucleic acid,as evidenced by a detectable signal, under conditions in which the probedoes not hybridize to other nucleic acids (e.g., animal cell or otherbacterial nucleic acids) present in the sample. A variety of factorsincluding the length and base composition of the probe, the extent ofbase mismatching between the probe and the target nucleic acid, thepresence of salt and organic solvents, probe concentration, and thetemperature affect hybridization, and optimal hybridization conditionsmust often be determined empirically. For discussions of nucleic acidprobe design and annealing conditions, see, for example, [81], supra,Ausubel, F., et al. [8] [ hereinafter referred to as Sambrook], Methodsin Enzymology [67] or Hybridization with Nucleic Acid Probes [42] all ofwhich are incorporated herein by reference.

Usually, at least a part of the probe will have considerable sequenceidentity with the target nucleic acid. Although the extent of thesequence identity required for specific hybridization will depend on thelength of the probe and the hybridization conditions, the probe willusually have at least 70% identity to the target nucleic acid, moreusually at least 80% identity, still more usually at least 90% identityand most usually at least 95% or 100% identity.

A probe can be identified as capable of hybridizing specifically to itstarget nucleic acid by hybridizing the probe to a sample treatedaccording the protocol of this invention where the sample contains bothtarget virus and animal cells (e.g., nerve cells). A probe is specificif the probe's characteristic signal is associated with the herpesvirusDNA in the sample and not generally with the DNA of the host cells andnon-biological materials (e.g., substrate) in a sample.

The following stringent hybridization and washing conditions will beadequate to distinguish a specific probe (e.g., a fluorescently labeledDNA probe) from a probe that is not specific: incubation of the probewith the sample for 12 hours at 37° C. in a solution containingdenatured probe, 50% formamide, 2×SSC, and 0.1% (w/v) dextran sulfate,followed by washing in 1×SSC at 70° C. for 5 minutes; 2×SSC at 37° C.for 5 minutes; 0.2×SSC at room temperature for 5 minutes, and H₂O atroom temperature for 5 minutes. Those of skill will be aware that itwill often be advantageous in nucleic acid hybridizations (i.e., insitu, Southern, or other) to include detergents (e.g., sodium dodecylsulfate), chelating agents (e.g., EDTA) or other reagents (e.g.,buffers, Denhardt's solution, dextran sulfate) in the hybridization orwash solutions. To test the specificity of the virus specific probes,the probes can be tested on host cells containing the KS-associatedherpesvirus and compared with the results from cells containingnon-KS-associated virus.

It will be apparent to those of ordinary skill in the art that aconvenient method for determining whether a probe is specific for aKS-associated viral nucleic acid utilizes a Southern blot (or Dot blot)using DNA prepared from one or more KS-associated human herpesviruses ofthe invention. Briefly, to identify a target specific probe DNA isisolated from the virus. Test DNA either viral or cellular istransferred to a solid (e.g., charged nylon) matrix. The probes arelabelled following conventional methods. Following denaturation and/orprehybridization steps known in the art, the probe is hybridized to theimmobilized DNAs under stringent conditions. Stringent hybridizationconditions will depend on the probe used and can be estimated from thecalculated T_(m) (melting temperature) of the hybridized probe (see,e.g., Sambrook for a description of calculation of the T_(m)). Forradioactively-labeled DNA or RNA probes an example of stringenthybridization conditions is hybridization in a solution containingdenatured probe and 5×SSC at 65° C. for 8-24 hours followed by washes in0.1×SSC, 0.1% SDS (sodium dodecyl sulfate) at 50-65° C. In general, thetemperature and salt concentration are chosen so that the posthybridization wash occurs at a temperature that is about 5° C. below theT_(M) of the hybrid. Thus for a particular salt concentration thetemperature may be selected that is 5° C. below the T_(M) or conversely,for a particular temperature, the salt concentration is chosen toprovide a T_(M) for the hybrid that is 5° C. warmer than the washtemperature. Following stringent hybridization and washing, a probe thathybridizes to the KS-associated viral DNA but not to the non-KSassociated viral DNA, as evidenced by the presence of a signalassociated with the appropriate target and the absence of a signal fromthe non-target nucleic acids, is identified as specific for the KSassociated virus. It is further appreciated that in determining probespecificity and in utilizing the method of this invention to detectKS-associated herpesvirus, a certain amount of background signal istypical and can easily be distinguished by one of skill from a specificsignal. Two fold signal over background is acceptable.

A preferred method for detecting the KS-associated herpesvirus is theuse of PCR and/or dot blot hybridization. The presence or absence of anKS agent for detection or prognosis, or risk assessment for KS includesSouthern transfers, solution hybridization or non-radioactive detectionsystems, all of which are well known to those of skill in the art.Hybridization is carried out using probes. Visualization of thehybridized portions allows the qualitative determination of the presenceor absence of the causal agent.

Similarly, a Northern transfer may be used for the detection of messagein samples of RNA or reverse transcriptase PCR and cDNA can be detectedby methods described above. This procedure is also well known in theart. See [81] incorporated by reference herein.

An alternative means for determining the presence of the humanherpesvirus is in situ hybridization, or more recently, in situpolymerase chain reaction. In situ PCR is described in Neuvo et al.[71], Intracellular localization of polymerase chain reaction(PCR)-amplified Hepatitis C cDNA; Bagasra et al. [10], Detection ofHuman Immunodeficiency virus type 1 provirus in mononuclear cells by insitu polymerase chain reaction; and Heniford et al. [35], Variation incellular EGF receptor mRNA expression demonstrated by in situ reversetranscriptase polymerase chain reaction. In situ hybridization assaysare well known and are generally described in Methods Enzymol. [67]incorporated by reference herein. In an in situ hybridization, cells arefixed to a solid support, typically a glass slide. The cells are thencontacted with a hybridization solution at a moderate temperature topermit annealing of target-specific probes that are labelled. The probesare preferably labelled with radioisotopes or fluorescent reporters.

The above described probes are also useful for in-situ hybridization orin order to locate tissues which express this gene, or for otherhybridization assays for the presence of this gene or its mRNA invarious biological tissues. In-situ hybridization is a sensitivelocalization method which is not dependent on expression of antigens ornative vs. denatured conditions.

Oligonucleotide (oligo) probes, synthetic oligonucleotide probes orriboprobes made from KSHV phagemids/plasmids, are relatively homogeneousreagents and successful hybridization conditions in tissue sections isreadily transferable from one probe to another. Commercially synthesizedoligonucleotide probes are prepared against the identified genes. Theseprobes are chosen for length (45-65 mers), high G-C content (50-70%) andare screened for uniqueness against other viral sequences in GenBank.

Oligonucleotides are 3′ end-labeled with [α-³⁵S]dATP to specificactivities in the range of 1×10¹⁰ dpm/ug using terminal deoxynucleotidyltransferase. Unincorporated labeled nucleotides are removed from theoligo probe by centrifugation through a Sephadex G-25 column or byelution from a Waters Sep Pak C-18 column.

KS tissue embedded in OCT compound and snap frozen in freezingisopentane cooled with dry ice is cut at 6 μm intervals and thawed onto3-aminopropyltriethoxysilane treated slides and allowed to air dry. Theslides are then be fixed in 4% freshly prepared paraformaldehyde, rinsedin water. Formalin-fixed, paraffin embedded KS tissues cut at 6 μm andbaked onto glass slides can also be used. The sections are thendeparaffinized in xylenes and rehydrated through graded alcohols.Prehybridization in 20 mM Tris Ph 7.5, 0.02% Denhardt's solution, 10%dextran sulfate for 30 min at 37° C. is followed by hybridizationovernight in a solution of 50% formamide (v/v), 10% dextran sulfate(w/v), 20 mM sodium phosphate (Ph 7.4), 3×SSC, 1×Denhardt's solution,100 ug/ml salmon sperm DNA, 125 ug/ml yeast tRNA and the oligo probe(10⁶ cpm/ml) at 42° C. overnight. The slides are washed twice with 2×SSCand twice with 1×SSC for 15 minutes each at room temperature andvisualized by autoradiography. Briefly, sections are dehydrated throughgraded alcohols containing 0.3M ammonium acetate and air dried. Theslides are dipped in Kodak NTB2 emulsion, exposed for days to weeks,developed, and counterstained with hematoxylin and eoxin. Alternativeimmunohistochemical protocols may be employed which are known to thoseskilled in the art.

IV. Treatment of Human Herpesvirus-Induced KS

This invention provides a method of treating a subject with Kaposi'ssarcoma, comprising administering to the subject an effective amount ofthe antisense molecule capable of hybridizing to the isolated DNAmolecule under conditions such that the antisense molecule selectivelyenters a tumor cell of the subject, so as to treat the subject.

This invention provides a method for treating a subject with Kaposi'ssarcoma (KS) comprising administering to the subject having a humanherpesvirus-associated KS a pharmaceutically effective amount of anantiviral agent in a pharmaceutically acceptable carrier, wherein theagent is effective to treat the subject with KS-associated human herpesvirus.

Further, this invention provides a method of prophylaxis or treatmentfor Kaposi's sarcoma (KS) by administering to a patient at risk for KS,an antibody that binds to the human herpesvirus in a pharmaceuticallyacceptable carrier. In one embodiment the antiviral drug is used totreat a subject with the DNA herpesvirus of the subject invention.

The use of combinations of antiviral drugs and sequential treatments areuseful for treatment of herpesvirus infections and will also be usefulfor the treatment of herpesvirus-induced KS. For example, Snoeck at al.[88], found additive or synergistic effects against CMV when combiningantiherpes drugs (e.g., combinations of zidovudine[3′-azido-3′-deoxythymidine, AZT] with HPMPC, ganciclovir, foscarnet oracyclovir or of HPMPC with other antivirals). Similarly, in treatment ofcytomegalovirus retinitis, induction with ganciclovir followed bymaintenance with foscarnet has been suggested as a way to maximizeefficacy while minimizing the adverse side effects of either treatmentalone. An anti-herpetic composition that contains acyclovir and, e.g.,2-acetylpyridine-5-((2-pyridylamino)thiocarbonyl)-thiocarbonohydrazoneis described in U.S. Pat. No. 5,175,165 (assigned to Burroughs WellcomeCo.). Combinations of TS-inhibitors and viral TK-inhibitors inantiherpetic medicines are disclosed in U.S. Pat. No. 5,137,724,assigned to Stichting Rega VZW. A synergistic inhibitory effect on EBVreplication using certain ratios of combinations of HPMPC with AZT wasreported by Lin et al. [56].

U.S. Pat. Nos. 5,164,395 and 5,021,437 (Blumenkopf; Burroughs Wellcome)describe the use of a ribonucleotide reductase inhibitor (anacetylpyridine derivative) for treatment of herpes infections, includingthe use of the acetylpyridine derivative in combination with acyclovir.U.S. Pat. No. 5,137,724 (Balzari et al. [11]) describes the use ofthymilydate synthase inhibitors (e.g., 5-fluoro-uracil and5-fluoro-2′-deoxyuridine) in combination with compounds having viralthymidine kinase inhibiting activity.

With the discovery of a disease causal agent for KS now identified,effective therapeutic or prophalactic protocols to alleviate or preventthe symptoms of herpes virus-associated KS can be formulated. Due to theviral nature of the disease, antiviral agents have application here fortreatment, such as interferons, nucleoside analogues, ribavirin,amantadine, and pyrophosphate analogues of phosphonoacetic acid(foscarnet) (reviewed in Gorbach, S. L., et al. [28]) and the like.Immunological therapy will also be effective in many cases to manage andalleviate symptoms caused by the disease agents described here.Antiviral agents include agents or compositions that directly bind toviral products and interfere with disease progress; and, excludes agentsthat do not impact directly on viral multiplication or viral titer.Antiviral agents do not include immunoregulatory agents that do notdirectly affect viral titer or bind to viral products. Antiviral agentsare effective if they inactivate the virus, otherwise inhibit itsinfectivity or multiplication, or alleviate the symptoms of KS.

A. Antiviral Agents.

The antiherpesvirus agents that will be useful for treatingvirus-induced KS can be grouped into broad classes based on theirpresumed modes of action. These classes include agents that act (i) byinhibition of viral DNA polymerase, (ii) by targeting other viralenzymes and proteins, (iii) by miscellaneous or incompletely understoodmechanisms, or (iv) by binding a target nucleic acid (i.e., inhibitorynucleic acid therapeutics). Antiviral agents may also be used incombination (i.e., together or sequentially) to achieve synergistic oradditive effects or other benefits.

Although it is convenient to group antiviral agents by their supposedmechanism of action, the applicants do not intend to be bound by anyparticular mechanism of antiviral action. Moreover, it will beunderstood by those of skill that an agent may act on more than onetarget in a virus or virus-infected cell or through more than onemechanism.

i) Inhibitors of Viral DNA Polymerase

Many antiherpesvirus agents in clinical use or in development today arenucleoside analogs believed to act through inhibition of viral DNAreplication, especially through inhibition of viral DNA polymerase.These nucleoside analogs act as alternative substrates for the viral DNApolymerase or as competitive inhibitors of DNA polymerase substrates.Usually these agents are preferentially phosphorylated by viralthymidine kinase (TK), if one is present, and/or have higher affinityfor viral DNA polymerase than for the cellular DNA polymerases,resulting in selective antiviral activity. Where a nucleoside analogueis incorporated into the viral DNA, viral activity or reproduction maybe affected in a variety of ways. For example, the analogue may act as achain terminator, cause increased lability (e.g., susceptibility tobreakage) of analogue-containing DNA, and/or impair the ability of thesubstituted DNA to act as template for transcription or replication(see, e.g., Balzarini et al. [11]).

It will be known to one of skill that, like many drugs, many of theagents useful for treatment of herpes virus infections are modified(i.e., “activated”) by the host, host cell, or virus-infected host cellmetabolic enzymes. For example, acyclovir is triphosphorylated to itsactive form, with the first phosphorylation being carried out by theherpes virus thymidine kinase, when present. Other examples are thereported conversion of the compound HOE 602 to ganciclovir in athree-step metabolic pathway (Winkler et al. [95]) and thephosphorylation of ganciclovir to its active form by, e.g., a CMVnucleotide kinase. It will be apparent to one of skill that the specificmetabolic capabilities of a virus can affect the sensitivity of thatvirus to specific drugs, and is one factor in the choice of an antiviraldrug. The mechanism of action of certain anti-herpesvirus agents isdiscussed in De Clercq [22] and in other references cited supra andinfra, all of which are incorporated by reference herein.

Anti-herpesvirus medications suitable for treating viral induced KSinclude, but are not limited to, nucleoside analogs including acyclicnucleoside phosphonate analogs (e.g., phosphonylmethoxyalkylpurines and-pyrimidines), and cyclic nucleoside analogs. These include drugs suchas: vidarabine (9-β-D-arabinofuranosyladenine; adenine arabinoside,ara-A, Vira-A, Parke-Davis); 1-β-D-arabinofuranosyluracil (ara-U);1-β-D-arabinofuranosyl-cytosine (ara-C); HPMPC[(S)-1-β-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine (e.g., GS 504Gilead Science)] and its cyclic form (cHPMPC); HPMPA[(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine] and its cyclic form(cHPMPA); (S)—HPMPDAP[(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine]; PMEDAP[9-(2-phosphonyl-methoxyethyl)-2,6-diaminopurine]; HOE 602[2-amino-9-(1,3-bis(isopropoxy)-2-propoxymethyl)purine]; PMEA[9-(2-phosphonylmethoxyethyl)adenine]; bromovinyl-deoxyuridine (Burnsand Sandford. [21]); 1-β-D-arabinofuranosyl-E-5-(2-bromovinyl)-uridineor -2′-deoxyuridine; BVaraU(1-β-D-arabinofuranosyl-E-5-(2-bromovinyl)-uracil, brovavir,Bristol-Myers Squibb, Yamsa Shoyu); BVDU[(E)-5-(2-bromovinyl)-2′-deoxyuridine, brivudin, e.g., Helpin] and itscarbocyclic analogue (in which the sugar moiety is replaced by acyclopentane ring); IVDU [(E)-5-(2-iodovinyl)-2′-deoxyuridine) and itscarbocyclic analogue, C-IVDU (Balzarini et al. [11])]; and 5-mercutithioanalogs of 2′-deoxyuridine (Holliday, J., and Williams, M. V. [38]);acyclovir [9-([2-hydroxyethoxy]methyl)guanine; e.g., Zovirax (BurroughsWellcome)]; penciclovir (9-[4-hydroxy-2-(hydroxymethyl)butyl]-guanine);ganciclovir [(9-[1,3-dihydroxy-2 propoxymethyl]-guanine) e.g., Cymevene,Cytovene (Syntex), DHPG (Stals et al. [89]]; isopropylether derivativesof ganciclovir (see, e.g., Winkelmann et al., [94]); cygalovir;famciclovir [2-amino-9-(4-acetoxy-3-(acetoxymethyl)but-1-yl)purine(Smithkline Beecham)]; valacyclovir (Burroughs Wellcome); desciclovir[(2-amino-9-(2-ethoxymethyl)purine)] and2-amino-9-(2-hydroxyethoxymethyl)-9H-purine, prodrugs of acyclovir]; CDG(carbocyclic 2′-deoxyguanosine); and purine nucleosides with thepentafuranosyl ring replaced by a cyclo butane ring (e.g., cyclobut-A[(+−)-9-[1β,2α,3β)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine],cyclobut-G[(+−)-9-[(1β,2α,3β)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine], BHCG[(R)-(1α,2β,1α)-9-(2,3-bis(hydroxymethyl)cyclobutyl]guanine], and anactive isomer of racemic BHCG, SQ 34,514[1R-1α,2β,3α)-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one(see, Braitman et al. (1991) [20]]. Certain of these antiherpesviralagents are discussed in Gorach at al. [28]; Saunders et al., [82];Yamanaka et al., [96]; Greenspan et al. [29], all of which areincorporated by reference herein.

Triciribine and triciribine monophosphate are potent inhibitors againstherpes viruses. (Ickes et al. [43], incorporated by reference herein),HIV-1 and HIV-2 (Kucera et al. [51], incorporated by reference herein)and are additional nucleoside analogs that may be used to treat KS. Anexemplary protocol for these agents is an intravenous injection of about0.35 mg/meter² (0.7 mg/kg) once weekly or every other week for at leasttwo doses, preferably up to about four to eight weeks.

Acyclovir and ganciclovir are of interest because of their accepted usein clinical settings. Acyclovir, an acyclic analogue of guanine, isphosphorylated by a herpesvirus thymidine kinase and undergoes furtherphosphorylation to be incorporated as a chain terminator by the viralDNA polymerase during viral replication. It has therapeutic activityagainst a broad range of herpesviruses, Herpes simplex Types 1 and 2,Varicella-Zoster, Cytomegalovirus, and Epstein-Barr Virus, and is usedto treat disease such as herpes encephalitis, neonatal herpesvirusinfections, chickenpox in immunocompromised hosts, herpes zosterrecurrences, CMV retinitis, EBV infections, chronic fatigue syndrome,and hairy leukoplakia in AIDS patients. Exemplary intravenous dosages ororal dosages are 250 mg/kg/m² body surface area, every 8 hours for 7days, or maintenance doses of 200-400 mg IV or orally twice a day tosuppress recurrence. Ganciclovir has been shown to be more active thanacyclovir against some herpesviruses. See, e.g., Oren and Soble [73].Treatment protocols for ganciclovir are 5 mg/kg twice a day IV or 2.5mg/kg three times a day for 10-14 days. Maintenance doses are 5-6 mg/kgfor 5-7 days.

Also of interest is HPMPC. HPMPC is reported to be more active thaneither acyclovir or ganciclovir in the chemotherapy and prophylaxis ofvarious HSV-1, HSV-2, TK-HSV, VZV or CMV infections in animal models([22], supra).

Nucleoside analogs such as BVaraU are potent inhibitors of HSV-1, EBV,and VZV that have greater activity than acyclovir in animal models ofencephalitis. FIAC (fluoroidoarbinosyl cytosine) and its relatedfluoroethyl and iodo compounds (e.g., FEAU, FIAU) have potent selectiveactivity against herpesviruses, and HPMPA((S)-1-([3-hydroxy-2-phosphorylmethoxy]propyl)adenine) has beendemonstrated to be more potent against HSV and CMV than acyclovir organciclovir and are of choice in advanced cases of KS. Cladribine(2-chlorodeoxyadenosine) is another nucleoside analogue known as ahighly specific antilymphocyte agent (i.e., a immunosuppressive drug).

Other useful antiviral agents include: 5-thien-2-yl-2′-deoxyuridinederivatives, e.g., BTDU [5-5(5-bromothien-2-yl)-2′-deoxyuridine] andCTDU [b-(5-chlorothien-2-yl)-2′-deoxyuridine]: and OXT-A[9-(2-deoxy-2-hydroxymethyl-β-D-erythro-oxetanosyl)adenine] and OXT-G[9-(2-deoxy-2-hydroxymethyl-O-D-erythro-oxetanosyl)guanine]. AlthoughOXT-G is believed to act by inhibiting viral DNA synthesis its mechanismof action has not yet been elucidated. These and other compounds aredescribed in Andrei et al. [5] which is incorporated by referenceherein. Additional antiviral purine derivatives useful in treatingherpesvirus infections are disclosed in U.S. Pat. No. 5,108,994(assigned to Beecham Group P.L.C.). 6-Methoxypurine arabinoside (ara-M,Burroughs Wellcome) is a potent inhibitor of varicella-zoster virus, andwill be useful for treatment of KS.

Certain thymidine analogs [e.g., idoxuridine (5-ido-2′-deoxyuridine)]and trifluorothymidine) have antiherpes viral activity, but due to theirsystemic toxicity, are largely used for topical herpesviral infections,including HSV stromal keratitis and uveitis, and are not preferred hereunless other options are ruled out.

Other useful antiviral agents that have demonstrated antiherpes viralactivity include foscarnet sodium (trisodium phosphonoformate, PFA,Foscavir (Astra)) and phosphonoacetic acid (PAA). Foscarnet is aninorganic pyrophosphate analogue that acts by competitively blocking thepyrophosphate-binding site of DNA polymerase. These agents which blockDNA polymerase directly without processing by viral thymidine kinase.Foscarnet is reported to be less toxic than PAA.

ii) Agents that Target Viral Proteins Other than DNA Polymerase or OtherViral Functions.

Although applicants do not intend to be bound by a particular mechanismof antiviral action, the antiherpes-virus agents described above arebelieved to act through inhibition of viral DNA polymerase. However,viral replication requires not only the replication of the viral nucleicacid but also the production of viral proteins and other essentialcomponents. Accordingly, the present invention contemplates treatment ofKS by the inhibition of viral proliferation by targeting viral proteinsother than DNA polymerase (e.g., by inhibition of their synthesis oractivity, or destruction of viral proteins after their synthesis). Forexample, administration of agents that inhibit a viral serine protease,e.g., such as one important in development of the viral capsid will beuseful in treatment of viral induced KS.

Other viral enzyme targets include: OMP decarboxylase inhibitors (atarget of, e.g., parazofurin), CTP synthetase inhibitors (targets of,e.g., cyclopentenylcytosine), IMP dehydrogenase, ribonucleotidereductase (a target of, e.g., carboxyl-containing N-alkyldipeptides asdescribed in U.S. Pat. No. 5,110,799 (Tolman et al., Merck)), thymidinekinase (a target of, e.g.,1-[2-(hydroxymethyl)cycloalkylmethyl]-5-substituted-uracils and-guanines as described in, e.g., U.S. Pat. Nos. 4,863,927 and 4,782,062(Tolman et al.; Merck)) as well as other enzymes. It will be apparent toone of ordinary skill in the art that there are additional viralproteins, both characterized and as yet to be discovered, that can serveas target for antiviral agents.

iv) Other Agents and Modes of Antiviral Action.

Kutapressin is a liver derivative available from Schwarz Parma ofMilwaukee, Wis. in an injectable form of 25 mg/ml. The recommendeddosage for herpesviruses is from 200 to 25 mg/ml per day for an averageadult of 150 pounds.

Poly(I) Poly(C₁₂U), an accepted antiviral drug known as Ampligen fromHEM Pharmaceuticals of Rockville, Md. has been shown to inhibitherpesviruses and is another antiviral agent suitable for treating KS.Intravenous injection is the preferred route of administration. Dosagesfrom about 100 to 600 mg/m² are administered two to three times weeklyto adults averaging 150 pounds. It is best to administer at least 200mg/m² per week.

Other antiviral agents reported to show activity against herpes viruses(e.g., varicella zoster and herpes simplex) and will be useful for thetreatment of herpesvirus-induced KS include mappicine ketone (SmithKlineBeecham); Compounds A,79296 and A,73209 (Abbott) for varicella zoster,and Compound 882C87 (Burroughs Wellcome) [see, The Pink Sheet 55(20) May17, 1993].

Interferon is known inhibit replication of herpes viruses. See [73],supra. Interferon has known toxicity problems and it is expected thatsecond generation derivatives will soon be available that will retaininterferon's antiviral properties but have reduced side affects.

It is also contemplated that herpes virus-induced KS may be treated byadministering a herpesvirus reactivating agent to induce reactivation ofthe latent virus. Preferably the reactivation is combined withsimultaneous or sequential administration of an anti-herpesvirus agent.Controlled reactivation over a short period of time or reactivation inthe presence of an antiviral agent is believed to minimize the adverseeffects of certain herpesvirus infections (e.g., as discussed in PCTApplication WO 93/04683). Reactivating agents include agents such asestrogen, phorbol esters, forskolin and β-adrenergic blocking agents.

Agents useful for treatment of herpesvirus infections and for treatmentof herpesvirus-induced KS are described in numerous U.S. patents. Forexample, ganciclovir is an example of a antiviral guanine acyclicnucleotide of the type described in U.S. Pat. Nos. 4,355,032 and4,603,219.

Acyclovir is an example of a class of antiviral purine derivatives,including 9-(2-hydroxyethylmethyl)adenine, of the type described in U.S.Pat. Nos. 4,287,188, 4,294,831 and 4,199,574.

Brivudin is an example of an antiviral deoxyuridine derivative of thetype described in U.S. Pat. No. 4,424,211.

Vidarabine is an example of an antiviral purine nucleoside of the typedescribed in British Pat. 1,159,290.

Brovavir is an example of an antiviral deoxyuridine derivative of thetype described in U.S. Pat. Nos. 4,542,210 and 4,386,076.

BHCG is an example of an antiviral carbocyclic nucleoside analogue ofthe type described in U.S. Pat. Nos. 5,153,352, 5,034,394 and 5,126,345.

HPMPC is an example of an antiviral phosphonyl methoxyalkyl derivativewith of the type described in U.S. Pat. No. 5,142,051.

CDG (Carbocyclic 2′-deoxyguanosine) is an example of an antiviralcarbocyclic nucleoside analogue of the type described in U.S. Pat. Nos.4,543,255, 4,855,466, and 4,894,458.

Foscarnet is described in U.S. Pat. No. 4,339,445.

Trifluridine and its corresponding ribonucleoside is described in U.S.Pat. No. 3,201,387.

U.S. Pat. No. 5,321,030 (Kaddurah-Daouk et al.; Amira) describes the useof creatine analogs as antiherpes viral agents. U.S. Pat. No. 5,306,722(Kim et al.; Bristol-Meyers Squibb) describes thymidine kinaseinhibitors useful for treating HSV infections and for inhibiting herpesthymidine kinase. Other antiherpesvirus compositions are described inU.S. Pat. Nos. 5,286,649 and 5,098,708 (Konishi et al., Bristol-MeyersSquibb) and 5,175,165 (Blumenkopf et al.; Burroughs Wellcome). U.S. Pat.No. 4,880,820 (Ashton et al.; Merck) describes the antiherpes virusagent (5)-9-(2,3-dihydroxy-1-propoxymethyl)guanine.

U.S. Pat. No. 4,708,935 (Suhadolnik et al.; Research Corporation)describes a 3′-deoxyadenosine compound effective in inhibiting HSV andEBV. U.S. Pat. No. 4,386,076 (Machida et al.; Yamasa Shoyu KabushikiKaisha) describes use of (E)-5-(2-halogenovinyl)-arabinofuranosyluracilas an antiherpesvirus agent. U.S. Pat. No. 4,340,599 (Lieb at al.; BayerAktiengesellschaft) describes phosphonohydroxyacetic acid derivativesuseful as antiherpes agents. U.S. Pat. Nos. 4,093,715 and 4,093,716 (Linet al. Research Corporation) describe 5′-amino-5′-deoxythymidine and5-iodo-5′-amino-2′,5′-dideoxycytidine as potent inhibitors of herpessimplex virus. U.S. Pat. No. 4,069,382 (Baker at al.; Parke, Davis &Company) describes 9-(5-O-Acyl-beta-D-arabinofuranosyladenine compoundsuseful as antiviral agents. U.S. Pat. No. 3,927,216 (Witkowski et al.)describes the use of 1,2,4-triazole-3-carboxamide and1,2,4-triazole-3-thiocarboxamide for inhibiting herpes virus infections.U.S. Pat. No. 5,179,093 (Afonso et al., Schering) describesquinoline-2,4-dione derivatives active against herpes simplex virus 1and 2, cytomegalovirus and Epstein Barr virus.

v) Inhibitory Nucleic Acid Therapeutics

Also contemplated here are inhibitory nucleic acid therapeutics whichcan inhibit the activity of herpesviruses in patients with KS.Inhibitory nucleic acids may be single-stranded nucleic acids, which canspecifically bind to a complementary nucleic acid sequence. By bindingto the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNAduplex or triplex is formed. These nucleic acids are often termed“antisense” because they are usually complementary to the sense orcoding strand of the gene, although recently approaches for use of“sense” nucleic acids have also been developed. The term “inhibitorynucleic acids” as used herein, refers to both “sense” and “antisense”nucleic acids.

By binding to the target nucleic acid, the inhibitory nucleic acid caninhibit the function of the target nucleic acid. This could, forexample, be a result of blocking DNA transcription, processing orpoly(A) addition to mRNA, DNA replication, translation, or promotinginhibitory mechanisms of the cells, such as promoting RNA degradation.Inhibitory nucleic acid methods therefore encompass a number ofdifferent approaches to altering expression of herpesvirus genes. Thesedifferent types of inhibitory nucleic acid technology are described inHelene, C. and Toulme, J. [34], which is hereby incorporated byreference and is referred to hereinafter as “Helene and Toulme.”

In brief, inhibitory nucleic acid therapy approaches can be classifiedinto those that target DNA sequences, those that target RNA sequences(including pre-mRNA and mRNA), those that target proteins (sense strandapproaches), and those that cause cleavage or chemical modification ofthe target nucleic acids.

Approaches targeting DNA fall into several categories. Nucleic acids canbe designed to bind to the major groove of the duplex DNA to form atriple helical or “triplex” structure. Alternatively, inhibitory nucleicacids are designed to bind to regions of single stranded DNA resultingfrom the opening of the duplex DNA during replication or transcription.See Helene and Toulme.

More commonly, inhibitory nucleic acids are designed to bind to mRNA ormRNA precursors. Inhibitory nucleic acids are used to prevent maturationof pre-mRNA. Inhibitory nucleic acids may be designed to interfere withRNA processing, splicing or translation.

The inhibitory nucleic acids can be targeted to mRNA. In this approach,the inhibitory nucleic acids are designed to specifically blocktranslation of the encoded protein. Using this approach, the inhibitorynucleic acid can be used to selectively suppress certain cellularfunctions by inhibition of translation of mRNA encoding criticalproteins. For example, an inhibitory nucleic acid complementary toregions of c-myc mRNA inhibits c-myc protein expression in a humanpromyelocytic leukemia cell line, HL60, which overexpresses the c-mycprotooncogene. See Wickstrom E. L., et al. [93] and Harel-Hellan, A., etal. [31A]. As described in Helene and Toulme, inhibitory nucleic acidstargeting mRNA have been shown to work by several different mechanismsto inhibit translation of the encoded protein(s).

The inhibitory nucleic acids introduced into the cell can also encompassthe “sense” strand of the gene or mRNA to trap or compete for theenzymes or binding proteins involved in mRNA translation. See Helene andToulme.

Lastly, the inhibitory nucleic acids can be used to induce chemicalinactivation or cleavage of the target genes or mRNA. Chemicalinactivation can occur by the induction of crosslinks between theinhibitory nucleic acid and the target nucleic acid within the cell.Other chemical modifications of the target nucleic acids induced byappropriately derivatized inhibitory nucleic acids may also be used.

Cleavage, and therefore inactivation, of the target nucleic acids may beeffected by attaching a substituent to the inhibitory nucleic acid whichcan be activated to induce cleavage reactions. The substituent can beone that affects either chemical, or enzymatic cleavage. Alternatively,cleavage can be induced by the use of ribozymes or catalytic RNA. Inthis approach, the inhibitory nucleic acids would comprise eithernaturally occurring RNA (ribozymes) or synthetic nucleic acids withcatalytic activity.

The targeting of inhibitory nucleic acids to specific cells of theimmune system by conjugation with targeting moieties binding receptorson the surface of these cells can be used for all of the above forms ofinhibitory nucleic acid therapy. This invention encompasses all of theforms of inhibitory nucleic acid therapy as described above and asdescribed in Helene and Toulme.

This invention relates to the targeting of inhibitory nucleic acids tosequences the human herpesvirus of the invention for use in treating KS.An example of an antiherpes virus inhibitory nucleic acid is ISIS 2922(ISIS Pharmaceuticals) which has activity against CMV [see,Biotechnology News 14(14) p. 5].

A problem associated with inhibitory nucleic acid therapy is theeffective delivery of the inhibitory nucleic acid to the target cell invivo and the subsequent internalization of the inhibitory nucleic acidby that cell. This can be accomplished by linking the inhibitory nucleicacid to a targeting moiety to form a conjugate that binds to a specificreceptor on the surface of the target infected cell, and which isinternalized after binding.

iii) Administration

The subjects to be treated or whose tissue may be used herein may be amammal, or more specifically a human, horse, pig, rabbit, dog, monkey,or rodent. In the preferred embodiment the subject is a human.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. Precise amountsof active ingredient required to be administered depend on the judgmentof the practitioner and are peculiar to each subject.

Suitable regimes for initial administration and booster shots are alsovariable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration.

As used herein administration means a method of administering to asubject. Such methods are well known to those skilled in the art andinclude, but are not limited to, administration topically, parenterally,orally, intravenously, intramuscularly, subcutaneously or by aerosol.Administration of the agent may be effected continuously orintermittently such that the therapeutic agent in the patient iseffective to treat a subject with Kaposi's sarcoma or a subject infectedwith a DNA virus associated with Kaposi's sarcoma.

The antiviral compositions for treating herpesvirus-induced KS arepreferably administered to human patients via oral, intravenous orparenteral administrations and other systemic forms. Those of skill inthe art will understand appropriate administration protocol for theindividual compositions to be employed by the physician.

The pharmaceutical formulations or compositions of this invention may bein the dosage form of solid, semi-solid, or liquid such as, e.g.,suspensions, aerosols or the like. Preferably the compositions areadministered in unit dosage forms suitable for single administration ofprecise dosage amounts. The compositions may also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological saline, Ringer's solution, dextrose solution, and Hank'ssolution. In addition, the pharmaceutical composition or formulation mayalso include other carriers, adjuvants; or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like. Effective amounts of suchdiluent or carrier are those amounts which are effective to obtain apharmaceutically acceptable formulation in terms of solubility ofcomponents, or biological activity, etc.

V. Immunological Approaches to Therapy.

Having identified a primary causal agent of KS in humans as a novelhuman herpesvirus, there are immunosuppressive therapies that canmodulate the immunologic dysfunction that arises from the presence ofviral infected tissue. In particular, agents that block theimmunological attack of the viral infected cells will ameliorate thesymptoms of KS and/or reduce the disease progress. Such therapiesinclude antibodies that specifically block the targeting of viralinfected cells. Such agents include antibodies which bind to cytokinesthat upregulate the immune system to target viral infected cells.

The antibody may be administered to a patient either singly or in acocktail containing two or more antibodies, other therapeutic agents,compositions, or the like, including, but not limited to,immuno-suppressive agents, potentiators and side-effect relievingagents. Of particular interest are immuno-suppressive agents useful insuppressing allergic reactions of a host. Immunosuppressive agents ofinterest include prednisone, prednisolone, DECADRON (Merck, Sharp &Dohme, West Point, Pa.), cyclophosphamide, cyclosporine,6-mercaptopurine, methotrexate, azathioprine and i.v. gamma globulin ortheir combination. Potentiators of interest include monensin, ammoniumchloride and chloroquine. All of these agents are administered ingenerally accepted efficacious dose ranges such as those disclosed inthe Physician Desk Reference, 41st Ed. (1987), Publisher Edward R.Barnhart, New Jersey.

Immune globulin from persons previously infected with humanherpesviruses or related viruses can be obtained using standardtechniques. Appropriate titers of antibodies are known for this therapyand are readily applied to the treatment of KS. Immune globulin can beadministered via parenteral injection or by intrathecal shunt. In brief,immune globulin preparations may be obtained from individual donors whoare screened for antibodies to the KS-associated human herpesvirus, andplasmas from high-titered donors are pooled. Alternatively, plasmas fromdonors are pooled and then tested for antibodies to the humanherpesvirus of the invention; high-titered pools are then selected foruse in KS patients.

Antibodies may be formulated into an injectable preparation. Parenteralformulations are known and are suitable for use in the invention,preferably for i.m. or i.v. administration. The formulations containingtherapeutically effective amounts of antibodies or immunotoxins areeither sterile liquid solutions, liquid suspensions or lyophilizedversions and optionally contain stabilizers or excipients. Lyophilizedcompositions are reconstituted with suitable diluents, e.g., water forinjection, saline, 0.3% glycine and the like, at a level of about from0.01 mg/kg of host body weight to 10 mg/kg where appropriate. Typically,the pharmaceutical compositions containing the antibodies orimmunotoxins will be administered in a therapeutically effective dose ina range of from about 0.01 mg/kg to about 5 mg/kg of the treated mammal.A preferred therapeutically effective dose of the pharmaceuticalcomposition containing antibody or immunotoxin will be in a range offrom about 0.01 mg/kg to about 0.5 mg/kg body weight of the treatedmammal administered over several days to two weeks by daily intravenousinfusion, each given over a one hour period, in a sequential patientdose-escalation regimen.

Antibody may be administered systemically by injection i.m.,subcutaneously or intraperitoneally or directly into KS lesions. Thedose will be dependent upon the properties of the antibody orimmunotoxin employed, e.g., its activity and biological half-life, theconcentration of antibody in the formulation, the site and rate ofdosage, the clinical tolerance of the patient involved, the diseaseafflicting the patient and the like as is well within the skill of thephysician.

The antibody of the present invention may be administered in solution.The pH of the solution should be in the range of pH 5 to 9.5, preferablypH 6.5 to 7.5. The antibody or derivatives thereof should be in asolution having a suitable pharmaceutically acceptable buffer such asphosphate, tris (hydroxymethyl) aminomethane-HCl or citrate and thelike. Buffer concentrations should be in the range of 1 to 100 mM. Thesolution of antibody may also contain a salt, such as sodium chloride orpotassium chloride in a concentration of 50 to 150 mM. An effectiveamount of a stabilizing agent such as an albumin, a globulin, a gelatin,a protamine or a salt of protamine may also be included and may be addedto a solution containing antibody or immunotoxin or to the compositionfrom which the solution is prepared.

Systemic administration of antibody is made daily, generally byintramuscular injection, although intravascular infusion is acceptable.Administration may also be intranasal or by other nonparenteral routes.Antibody or immunotoxin may also be administered via microspheres,liposomes or other microparticulate delivery systems placed in certaintissues including blood.

In therapeutic applications, the dosages of compounds used in accordancewith the invention vary depending on the class of compound and thecondition being treated. The age, weight, and clinical condition of therecipient patient; and the experience and judgment of the clinician orpractitioner administering the therapy are among the factors affectingthe selected dosage. For example, the dosage of an immunoglobulin canrange from about 0.1 milligram per kilogram of body weight per day toabout 10 mg/kg per day for polyclonal antibodies and about 5% to about20% of that amount for monoclonal antibodies. In such a case, theimmunoglobulin can be administered once daily as an intravenousinfusion. Preferably, the dosage is repeated daily until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. Generally, the dose should be sufficient totreat or ameliorate symptoms or signs of KS without producingunacceptable toxicity to the patient.

An effective amount of the compound is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Thedosing range varies with the compound used, the route of administrationand the potency of the particular compound.

VI. Vaccines and Prophylaxis for KS

This invention provides a method of vaccinating a subject againstKaposi's sarcoma, comprising administering to the subject an effectiveamount of the peptide or polypeptide encoded by the isolated DNAmolecule, and a suitable acceptable carrier, thereby vaccinating thesubject. In one embodiment naked DNA is administering to the subject inan effective amount to vaccinate a subject against Kaposi's sarcoma.

This invention provides a method of immunizing a subject against adisease caused by the DNA herpesvirus associated with Kaposi's sarcomawhich comprises administering to the subject an effective immunizingdose of the isolated herpesvirus vaccine.

A. Vaccines

The invention also provides substances suitable for use as vaccines forthe prevention of KS and methods for administering them. The vaccinesare directed against the human herpesvirus of the invention, and mostpreferably comprise antigen obtained from the KS-associated humanherpesvirus.

Vaccines can be made recombinantly. Typically, a vaccine will includefrom about 1 to about 50 micrograms of antigen or antigenic protein orpeptide. More preferably, the amount of protein is from about 15 toabout 45 micrograms. Typically, the vaccine is formulated so that a doseincludes about 0.5 milliliters. The vaccine may be administered by anyroute known in the art. Preferably, the route is parenteral. Morepreferably, it is subcutaneous or intramuscular.

There are a number of strategies for amplifying an antigen'seffectiveness, particularly as related to the art of vaccines. Forexample, cyclization or circularization of a peptide can increase thepeptide's antigenic and immunogenic potency. See U.S. Pat. No. 5,001,049which is incorporated by reference herein. More conventionally, anantigen can be conjugated to a suitable carrier, usually a proteinmolecule. This procedure has several facets. It can allow multiplecopies of an antigen, such as a peptide, to be conjugated to a singlelarger carrier molecule. Additionally, the carrier may possessproperties which facilitate transport, binding, absorption or transferof the antigen.

For parenteral administration, such as subcutaneous injection, examplesof suitable carriers are the tetanus toxoid, the diphtheria toxoid,serum albumin and lamprey, or keyhole limpet, hemocyanin because theyprovide the resultant conjugate with minimum genetic restriction.Conjugates including these universal carriers can function as T cellclone activators in individuals having very different gene sets.

The conjugation between a peptide and a carrier can be accomplishedusing one of the methods known in the art. Specifically, the conjugationcan use bifunctional cross-linkers as binding agents as detailed, forexample, by Means and Feeney, “A recent review of protein modificationtechniques,” Bioconjugate Chem. 1:2-12 (1990).

Vaccines against a number of the Herpesviruses have been successfullydeveloped. Vaccines against Varicella-Zoster Virus using a liveattenuated Oka strain is effective in preventing herpes zoster in theelderly, and in preventing chickenpox in both immunocompromised andnormal children (Hardy, I., et al. [30]; Hardy, I. et al. [31]; Levin,M. J. et al. [54]; Gershon, A. A. [26]. Vaccines against Herpes simplexTypes 1 and 2 are also commercially available with some success inprotection against primary disease, but have been less successful inpreventing the establishment of latent infection in sensory ganglia(Roizman, B. [78]; Skinner, G. R. et al. [87]).

Vaccines against the human herpesvirus can be made by isolatingextracellular viral particles from infected cell cultures, inactivatingthe virus with formaldehyde followed by ultracentrifugation toconcentrate the viral particles and remove the formaldehyde, andimmunizing individuals with 2 or 3 doses containing 1×10⁹ virusparticles (Skinner, G. R. et al. [86]). Alternatively, envelopeglycoproteins can be expressed in E. coli or transfected into stablemammalian cell lines, the proteins can be purified and used forvaccination (Lasky, L. A. [53]). MHC-binding peptides from cellsinfected with the human herpesvirus can be identified for vaccinecandidates per the methodology of [61], supra.

The antigen may be combined or mixed with various solutions and othercompounds as is known in the art. For example, it may be administered inwater, saline or buffered vehicles with or without various adjuvants orimmunodiluting agents. Examples of such adjuvants or agents includealuminum hydroxide, aluminum phosphate, aluminum potassium sulfate(alum), beryllium sulfate, silica, kaolin, carbon, water-in-oilemulsions, oil-in-water emulsions, muramyl dipeptide, bacterialendotoxin, lipid X, Corynebacterium parvum (Propionibacterium acnes),Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin,lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran,blocked copolymers or other synthetic adjuvants. Such adjuvants areavailable commercially from various sources, for example, Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.) or Freund's IncompleteAdjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.).Other suitable adjuvants are Amphigen (oil-in-water), Alhydrogel(aluminum hydroxide), or a mixture of Amphigen and Alhydrogel. Onlyaluminum is approved for human use.

The proportion of antigen and adjuvant can be varied over a broad rangeso long as both are present in effective amounts. For example, aluminumhydroxide can be present in an amount of about 0.5% of the vaccinemixture (Al₂O, basis). On a per-dose basis, the amount of the antigencan range from about 0.1 μg to about 100 μg protein per patient. Apreferable range is from about 1 μg to about 50 μg per dose. A morepreferred range is about 15 μg to about 45 μg. A suitable dose size isabout 0.5 ml. Accordingly, a dose for intramuscular injection, forexample, would comprise 0.5 ml containing 45 μg of antigen in admixturewith 0.5% aluminum hydroxide. After formulation, the vaccine may beincorporated into a sterile container which is then sealed and stored ata low temperature, for example 4° C., or it may be freeze-dried.Lyophilization permits long-term storage in a stabilized form.

The vaccines may be administered by any conventional method for theadministration of vaccines including oral and parenteral (e.g.,subcutaneous or intramuscular) injection. Intramuscular administrationis preferred. The treatment may consist of a single dose of vaccine or aplurality of doses over a period of time. It is preferred that the dosebe given to a human patient within the first 8 months of life. Theantigen of the invention can be combined with appropriate doses ofcompounds including influenza antigens, such as influenza type Aantigens. Also, the antigen could be a component of a recombinantvaccine which could be adaptable for oral administration.

Vaccines of the invention may be combined with other vaccines for otherdiseases to produce multivalent vaccines. A pharmaceutically effectiveamount of the antigen can be employed with a pharmaceutically acceptablecarrier such as a protein or diluent useful for the vaccination ofmammals, particularly humans.

Other vaccines may be prepared according to methods well-known to thoseskilled in the art.

Those of skill will readily recognize that it is only necessary toexpose a mammal to appropriate epitopes in order to elicit effectiveimmunoprotection. The epitopes are typically segments of amino acidswhich are a small portion of the whole protein. Using recombinantgenetics, it is routine to alter a natural protein's primary structureto create derivatives embracing epitopes that are identical to orsubstantially the same as (immunologically equivalent to) the naturallyoccurring epitopes. Such derivatives may include peptide fragments,amino acid substitutions, amino acid deletions and amino acid additionsof the amino acid sequence for the viral proteins from the humanherpesvirus. For example, it is known in the protein art that certainamino acid residues can be substituted with amino acids of similar sizeand polarity without an undue effect upon the biological activity of theprotein. The human herpesvirus proteins have significant tertiarystructure and the epitopes are usually conformational. Thus,modifications should generally preserve conformation to produce aprotective immune response.

B. Antibody Prophylaxis

Therapeutic, intravenous, polyclonal or monoclonal antibodies can beenused as a mode of passive immunotherapy of herpesviral diseasesincluding perinatal varicella and CMV. Immune globulin from personspreviously infected with the human herpesvirus and bearing a suitablyhigh titer of antibodies against the virus can be given in combinationwith antiviral agents (e.g. ganciclovir), or in combination with othermodes of immunotherapy that are currently being evaluated for thetreatment of KS, which are targeted to modulating the immune response(i.e. treatment with copolymer-1, antiidiotypic monoclonal antibodies, Tcell “vaccination”). Antibodies to human herpesvirus can be administeredto the patient as described herein. Antibodies specific for an epitopeexpressed on cells infected with the human herpesvirus are preferred andcan be obtained as described above.

A polypeptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

C. Monitoring Therapeutic Efficacy

This invention provides a method for monitoring the therapeutic efficacyof treatment for Kaposi's sarcoma, which comprises determining in afirst sample from a subject with Kaposi's sarcoma the presence of theisolated DNA molecule, administering to the subject a therapeutic amountof an agent such that the agent is contacted to the cell in a sample,determining after a suitable period of time the amount of the isolatedDNA molecule in the second sample from the treated subject, andcomparing the amount of isolated DNA molecule determined in the firstsample with the amount determined in the second sample, a differenceindicating the effectiveness of the agent, thereby monitoring thetherapeutic efficacy of treatment for Kaposi's sarcoma. As definedherein “amount” is viral load or copy number. Methods of determiningviral load or copy number are known to those skilled in the art.

VII. Screening Assays for Pharmaceutical Agents of Interest inAlleviating the Symptoms of KS.

Since an agent involved in the causation or progression of KS has beenidentified and described here, assays directed to identifying potentialpharmaceutical agents that inhibit the biological activity of the agentare possible. KS drug screening assays which determine whether or not adrug has activity against the virus described herein are contemplated inthis invention. Such assays comprise incubating a compound to beevaluated for use in KS treatment with cells which express the KSassociated human herpesvirus proteins or peptides and determiningtherefrom the effect of the compound on the activity of such agent. Invitro assays in which the virus is maintained in suitable cell cultureare preferred, though in vivo animal models would also be effective.

Compounds with activity against the agent of interest or peptides fromsuch agent can be screened in in vitro as well as in vivo assay systems.In vitro assays include infecting peripheral blood leukocytes orsusceptible T cell lines such as MT-4 with the agent of interest in thepresence of varying concentrations of compounds targeted against viralreplication, including nucleoside analogs, chain terminators, antisenseoligonucleotides and random polypeptides (Asada, H. et al. [7]; Kikutaet al., [48] both incorporated by reference herein). Infected culturesand their supernatants can be assayed for the total amount of virusincluding the presence of the viral genome by quantitative PCR, by dotblot assays, or by using immunologic methods. For example, a culture ofsusceptible cells could be infected with the human herpesvirus in thepresence of various concentrations of drug, fixed on slides after aperiod of days, and examined for viral antigen by indirectimmunofluorescence with monoclonal antibodies to viral peptides ([48],supra. Alternatively, chemically adhered MT-4 cell monolayers can beused for an infectious agent assay using indirect immunofluorescentantibody staining to search for focus reduction (Higashi, K. et al.,[36], incorporated by reference herein).

As an alternative to whole cell in vitro assays, purified enzymesisolated from the human herpesvirus can be used as targets for rationaldrug design to determine the effect of the potential drug on enzymeactivity, such as thymidine phosphotransferase or DNA polymerase. Thegenes for these two enzymes are provided herein. A measure of enzymeactivity indicates effect on the agent itself.

Drug screens using herpes viral products are known and have beenpreviously described in EP 0514830 (herpes proteases) and WO 94/04920(U_(L)13 gene product).

This invention provides an assay for screening anti-KSchemotherapeutics. Infected cells can be incubated in the presence of achemical agent that is a potential chemotherapeutic against KS (e.g.acyclo-guanosine). The level of virus in the cells is then determinedafter several days by IFA for antigens or Southern blotting for viralgenome or Northern blotting for mRNA and compared to control cells. Thisassay can quickly screen large numbers of chemical compounds that may beuseful against KS.

Further, this invention provides an assay system that is employed toidentify drugs or other molecules capable of binding to the DNA moleculeor proteins, either in the cytoplasm or in the nucleus, therebyinhibiting or potentiating transcriptional activity. Such assay would beuseful in the development of drugs that would be specific againstparticular cellular activity, or that would potentiate such activity, intime or in level of activity.

This invention is further illustrated in the Experimental Detailssection which follows. This section is set forth to aid in anunderstanding of the invention but is not intended to, and should not beconstrued to, limit in any way the invention as set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS SECTION I Experiment 1 Representational DifferenceAnalysis (RDA) to Identify and Characterize Unique DNA Sequences in KSTissue

To search for foreign DNA sequences belonging to an infectious agent inAIDS-KS, representational difference analysis (RDA) was employed toidentify and characterize unique DNA sequences in KS tissue that areeither absent or present in low copy number in non-diseased tissueobtained from the same patient [58]. This method can detect adenovirusgenome added in single copy to human DNA but has not been used toidentify previously uncultured infectious agents. RDA is performed bymaking simplified “representations” of genomes from diseased and normaltissues from the same individual through PCR amplification of shortrestriction fragments. The DNA representation from the diseased tissueis then ligated to a priming sequence and hybridized to an excess ofunligated, normal tissue DNA representation. Only unique sequences foundin the diseased tissue have priming sequences on both DNA strands andare preferentially amplified during subsequent rounds of PCRamplification. This process can be repeated using different ligatedpriming sequences to enrich the sample for unique DNA sequences that areonly found in the tissue of interest.

DNA (10 μg) extracted from both the KS lesion and unaffected tissue wereseparately digested to completion with Bam HI (20 units/μg) at 37° C.for 2 hours and 2 μg of digestion fragments were ligated to NBam12 andNBam24 priming sequences [primer sequences described in 58]. Thirtycycles of PCR amplification were performed to amplify “representations”of both genomes. After construction of the genomic representations, KStester amplicons between 150 and 1500 bp were isolated from an agarosegel and NBam priming sequences were removed by digestion with NBam HI.To search for unique DNA sequences not found in non-KS driver DNA, asecond set of priming sequences (JBam12 and JBam24) was ligated ontoonly the KS tester DNA amplicons (FIG. 1, lane 1). 0.2 μg of ligated KSlesion amplicons were hybridized to 20 μg of unligated, normal tissuerepresentational amplicons. An aliquot of the hybridization product wasthen subjected to 10 cycles of PCR amplification using JBam24, followedby mung bean nuclease digestion. An aliquot of the mung bean-treateddifference product was then subjected to 15 more cycles of PCR with theJBam24 primer (FIG. 1, lane 2). Amplification products were redigestedwith Bam HI and 200 ng of the digested product was ligated to RBam12 andRBam24 primer sets for a second round of hybridization and PCRamplification (FIG. 1, lane 3). This enrichment procedure was repeated athird time using the JBam primer set (FIG. 1, lane 4). Both the originaldriver and the tester DNA samples (Table 2, Patient A) were subsequentlyfound to contain the AIDS-KS specific sequences KS330Bam and KS631Bam(previously identified as KS627Bam) indicating that RDA can besuccessfully employed when the target sequences are present in unequalcopy number in both tissues.

The initial round of DNA amplification-hybridization from KS and normaltissue resulted in a diffuse banding pattern (FIG. 1, lane 2), but fourbands at approximately 380, 450, 540 and 680 bp were identifiable afterthe second amplification-hybridization (FIG. 1, lane 3). These bandsbecame discrete after a third round of amplification-hybridization (FIG.1, lane 4). Control RDA, performed by hybridizing DNA extracted fromAIDS-KS tissue against itself, produced a single band at approximately540 bp (FIG. 1, lane 5). The four KS-associated bands (designatedKS330Bam, KS390Bam, KS480Bam, KS627Bam after digestion of the twoflanking 28 bp ligated priming sequences with Bam HI) were gel purifiedand cloned by insertion into the pCRII vector. PCR products were clonedin the pCRII vector using the TA cloning system (Invitrogen Corporation,San Diego, Calif.).

Experiment 2 Determination of the Specificity of AIDS-KS UniqueSequences

To determine the specificity of these sequences for AIDS-KS,random-primed ³²P-labeled inserts were hybridized to Southern blots ofDNA extracted from cryopreserved tissues obtained from patients with andwithout AIDS. All AIDS-KS specimens were examined microscopically formorphologic confirmation of KS and immunohistochemically for FactorVIII, Ulex europaeus and CD34 antigen expression. One of the AIDS-KSspecimens was apparently mislabeled since KS tissue was not detected onmicroscopic examination but was included in the KS specimen group forpurposes of statistical analysis. Control tissues used for comparison tothe KS lesions included 56 lymphomas from patients with and withoutAIDS, 19 hyperplastic lymph nodes from patients with and without AIDS, 5vascular tumors from nonAIDS patients and 13 tissues infected withopportunistic infections that commonly occur in AIDS patients. ControlDNA was also extracted from a consecutive series of 49 surgical biopsyspecimens from patients without AIDS. Additional clinical anddemographic information on the specimens was not collected to preservepatient confidentiality.

The tissues, listed in Table 1, were collected from diagnostic biopsiesand autopsies between 1983 and 1993 and stored at −70° C. Each tissuesample was from a different patient, except as noted in Table 1. Most ofthe 27 KS specimens were from lymph nodes dissected under surgicalconditions which diminishes possible contamination with normal skinflora. All specimens were digested with Bam HI prior to hybridization.

KS390Bam and KS480Bam hybridized nonspecifically to both KS and non-KStissues and were not further characterized. 20 of 27 (74%) AIDS-KS DNAshybridized with variable intensity to both KS330Bam and KS627Bam, andone additional KS specimen hybridized only to KS627Bam by Southernblotting (FIG. 2 and Table 1). In contrast to AIDS-KS lesions, only 6 of39 (15%) non-KS tissues from patients with AIDS hybridized to theKS330Bam and KS627Bam inserts (Table 1).

Specific hybridization did not occur with lymphoma or lymph node DNAfrom 36 persons without AIDS or with control DNA from 49 tissue biopsyspecimens obtained from a consecutive series of patients. DNA extractedfrom several vascular tumors, including a hemangiopericytoma, twoangiosarcomas and a lymphangioma, were also negative by Southern blothybridization. DNA extracted from tissues with opportunistic infectionscommon to AIDS patients, including 7 acid-fast bacillus (undeterminedspecies), cytomegalovirus, 1 cat-scratch bacillus, 2 cryptococcus and 1toxoplasmosis infected tissues, were negative by Southern blothybridization to KS330Bam and KS627Bam (Table 1).

TABLE 1 Southern blot hybridization for KS330Bam and KS627Bam and PCRamplification for KS330₂₃₄ in human tissues from individual patients.KS330Bam KS627Bam KS330₂₃₄ Southern hybrid- Southern hybrid- PCR Tissuen ization n(%) ization n(%) positive AIDS-KS 27* 20 (74) 21 (78) 25 (93)AIDS 27† 3 (11) 3 (11) 3 (11) lymphomas AIDS 12 3 (25) 3 (25) 3 (25)lymph nodes Non-AIDS 29 0 (0) 0 (0) 0 (0) Lymphomas Non-AIDS  7 0 (0) 0(0) 0 (0) lymph nodes Vascular  4§ 0 (0) 0 (0) 0 (0) tumors Opportu- 13π0 (0) 0 (0) 0 (0) nistic infections Consecutive 49¶** 0 (0) 0 (0) 0 (0)surgical biopsies Legend to Table 1: *Includes one AIDS-KS specimenunamplifiable for p53 exon 6 and one tissue which on microscopicexamination did not have any detectable KS tissue present. Both of thesesamples were negative by Southern blot hybridization to KS330Bam andKS627Bam and by PCR amplification for the KS330₂₃₄ amplicon. †Includes 7small non-cleaved cell lymphomas, 20 diffuse large cell andimmunoblastic lymphomas. Three of the lymphomas with immunoblasticmorphology were positive for KS330Bam and KS627Bam. ‡Includes 13anaplastic large cell lymphomas, 4 diffuse large cell lymphomas, 4 smalllymphocytic lymphomas/chronic lymphocytic leukemias, 3 hairy cellleukemias, 2 monocytoid B-cell lymphomas, 1 follicular small cleavedcell lymphoma, 1 Burkitt's lymphoma, 1 plasmacytoma. §Includes 2angiosarcomas, 1 hemangiopericytoma and 1 lymphangioma. πIncludes 2cryptococcus, 1 toxoplasmosis, 1 cat-scratch bacillus, 1cytomegalovirus, 1 Epstein-Barr virus, and 7 acid-fast bacillus infectedtissues. In addition, pure cultures of Mycobacterium avium-complex werenegative by Southern hybridization and PCR, and pure cultures ofMycoplasma penetrans were negative by PCR. ¶Tissues included skin,appendix, kidney, prostate, hernia sac, lung, fibrous tissue,gallbladder, colon, foreskin, thyroid, small bowel, adenoid, vein,axillary tissue, lipoma, heart, mouth, hemorrhoid, pseudoaneurysm andfistula track. Tissues were collected from a consecutive series ofbiopsies on patients without AIDS but with unknown HIV serostatus.**Apparent nonspecific hybridization at approximately 20 Kb occurred in4 consecutive surgical biopsy DNA samples: one colon and one hernia sacDNA sample hybridized to KS330Bam alone, another hernia sac DNA samplehybridized to KS627Bam alone and one appendix DNA sample hybridized toboth KS330Bam and KS627Bam. These samples did not hybridize in the330-630 bp range expected for these sequences and were PCR negative forKS330₂₃₄.

In addition, DNA from Epstein-Barr virus-infected peripheral bloodlymphocytes and pure cultures of Mycobacterium avium-complex were alsonegative by Southern hybridization. Overall, 20 of 27 (74%) AIDS-KSspecimens hybridized to KS330Bam and 21 of 27 (78%) AIDS-KS specimenshybridized to KS627Bam, compared to only 6 of 142 (4%) non-KS human DNAcontrol specimens (χ²=85.02, p<10⁻⁷ and χ²=92.4, p<10⁻⁷ respectively).

The sequence copy number in the AIDS-KS tissues was estimated bysimultaneous hybridization with KS330Bam and a 440 bp probe for theconstant region of the T cell receptor β gene [76]. Samples in lanes 5and 6 of FIGS. 2A-2B showed similar intensities for the two probesindicating an average copy number of approximately two KS330Bamsequences per cell, while remaining tissues had weaker hybridizationsignals for the KS330Bam probe.

Experiment 3 Characterization of KS330Bam and KS627Bam

To further characterize KS330Bam and KS627Bam, six clones for eachinsert were sequenced. The Sequenase version 2.0 (United StatesBiochemical, Cleveland, Ohio) system was used and sequencing wasperformed according to manufacturer's instructions. Nucleotidessequences were confirmed with an Applied Biosystems 373A Sequencer inthe DNA Sequencing Facilities at Columbia University.

KS330Bam is a 330 bp sequence with 51% G:C content (FIG. 3B) andKS627Bam is a 627 bp sequence with a 63% G:C content (FIG. 3C). KS330Bamhas 54% nucleotide identity to the BDLF1 open reading frame (ORF) ofEpstein-Barr virus (EBV). Further analysis revealed that both KS330Bamand KS627Bam code for amino acid sequences with homology to polypeptidesof viral origin. SwissProt and PIR protein databases were searched forhomologous ORF using BLASTX [3].

KS330Bam is 511 identical by amino acid homology to a portion of theORF26 open reading frame encoding the capsid protein VP23 (NCBI g.i.60348, bp 46024-46935) of herpesvirus saimiri [2], a gammaherpesviruswhich causes fulminant lymphoma in New world monkeys. This fragment alsohas a 39% identical amino acid sequence to the theoretical proteinencoded by the homologous open reading frame BDLF1 in EBV (NCBI g.i.59140, bp 132403-133307) [9]. The amino acid sequence encoded byKS627Bam is homologous with weaker identity (31%) to the tegumentprotein, gp140 (ORF 29, NCBI g.i. 60396, bp108782-112681) of herpesvirussaimiri.

Sequence data from KS330Bam was used to construct PCR primers to amplifya 234 bp fragment designated KS330₂₃₄ (FIG. 3B). The conditions for PCRanalyses were as follows: 94° C. for 2 min (1 cycle); 94° C. for 1 min,58° C. for 1 min, 72° C. for 1 min (35 cycles); 72° C. extension for 5min (1 cycle). Each PCR reaction used 0.1 μg of genomic DNA, 50 pmolesof each primer, 1 unit of Taq polymerase, 100 μM of each deoxynucleotidetriphosphate, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), and 0.1% Triton-X-100in a final volume of 25 μl. Amplifications were carried out in aPerkin-Elmer 480 Thermocycler with 1-s ramp times between steps.

Although Southern blot hybridization detected the KS330Bam sequence inonly 20 of 27 KS tissues, 25 of the 27 tissues were positive by PCRamplification for KS330₂₃₄ (FIGS. 4A-4B) demonstrating that KS330Bam ispresent in some KS lesions at levels below the threshold for detectionby Southern blot hybridization. All KS330₂₃₄ PCR products hybridized toa ³²P end-labelled 25 bp internal oligomer, confirming the specificityof the PCR (FIG. 4B). Of the two AIDS-KS specimens negative forKS330₂₃₄, both specimens appeared to be negative for technical reasons:one had no microscopically detectable KS tissue in the frozen sample(FIGS. 4A-4B, lane 3), and the other (FIGS. 4A-4B, lane 15) was negativein the control PCR amplification for the p53 gene indicating either DNAdegradation or the presence of PCR inhibitors in the sample. PCRamplification of the p53 tumor suppressor gene was used as a control forDNA quality. Sequences of p53 primers fromP6-5,5′-ACAGGGCTGGTTGCCCAGGGT-3′(SEQ ID No: 44); and P6-3.5′-AGTTGCAAACCAGACCTCAG-3′(SEQ ID NO: 45) [25].

Except for the 6 control samples from AIDS patients that were alsopositive by Southern blot hybridization, none of the other 136 controlspecimens were positive by PCR for KS330₂₃₄. All of these specimens wereamplifiable for the p53 gene, indicating that inadequate PCRamplification was not the reason for lack of detection of KS330₂₃₄ inthe control tissues. Samples containing DNA from two candidate KSagents, EBV and Mycoplasma penetrans (ATCC Accession No. 55252), apathogen commonly found in the genital tract of patients with AIDS-KS[59] were also negative for amplification of KS330₂₃₄. In addition,several KS specimens were tested using commercial PCR primers(Stratagene, La Jolla, Calif.) specific for mycoplasmata and primersspecific for the EBNA-2, EBNA-3C and EBER regions of EBV and werenegative [57].

Overall, DNA from 25 (93%) of 27 AIDS-KS tissues were positive by PCRcompared with DNA from 6 (4%) of 142 control tissues, including 6 (15%)of 39 non-KS lymph nodes and lymphomas from AIDS patients (χ²=38.2,p<10⁻⁶), 0 of 36 lymph nodes and lymphomas from nonAIDS patients(χ²=55.2, p<10⁻⁷) and 0 of 49 consecutive biopsy specimens (χ²=67.7,p<10⁻⁷). Thus, KS330₂₃₄ was found in all 25 amplifiable tissues withmicroscopically detectable AIDS-KS, but rarely occurred in non-KStissues, including tissues from AIDS patients.

Of the six control tissues from AIDS patients that were positive by bothPCR and Southern hybridization, two patients had KS elsewhere, two didnot develop KS and complete clinical histories for the remaining twopatients were unobtainable. Three of the six positive non-KS tissueswere lymph nodes with follicular hyperplasia taken from patients withAIDS. Given the high prevalence of KS among patients with AIDS, it ispossible that undetected microscopic foci of KS were present in theselymph nodes. The other three positive tissue specimens were B cellimmunoblastic lymphomas from AIDS patients. It is possible that theputative KS agent is also a cofactor for a subset of AIDS-associatedlymphomas [16, 17, 80].

To determine whether KS330Bam and KS627Bam are portions of a largergenome and to determine the proximity of the two sequences to eachother, samples of KS DNA were digested with Pvu II restriction enzymes.Digested genomic DNA from three AIDS-KS samples were hybridized toKS330Bam and KS627Bam by Southern blotting (FIG. 5). These sequenceshybridized to various sized fragments of the digested KS DNA indicatingthat both sequences are fragments of larger genomes. Differences in theKS330Bam hybridization pattern to Pvu II digests of the three AIDS-KSspecimens indicate that polymorphisms may occur in the larger genome.Individual fragments from the digests failed to simultaneously hybridizewith both KS330Bam and KS627Bam, demonstrating that these two Bam HIrestriction fragments are not adjacent to one another.

If KS330Bam and KS627Bam are heritable polymorphic DNA markers for KS,these sequences should be uniformly detected at non-KS tissue sites inpatients with AIDS-KS. Alternatively, if KS330Bam and KS627Bam aresequences specific for an exogenous infectious agent, it is likely thatsome tissues are uninfected and lack detectable KS330Bam and KS627Bamsequences. DNA extracted from multiple uninvolved tissues from threepatients with AIDS-KS were hybridized to ³²P-labelled KS330Bam andKS627Bam probes as well as analyzed by PCR using the KS330₂₃₄ primers(Table 2). While KS lesion DNA samples were positive for both bands,unaffected tissues were frequently negative for these sequences. KSlesions from patients A, B and C, and uninvolved skin and muscle frompatient A were positive for KS330Bam and KS627Bam, but muscle and braintissue from patient B and muscle, brain, colon, heart and hilar lymphnode tissues from patient C were negative for these sequences.Uninvolved stomach lining adjacent to the KS lesion in patient C waspositive by PCR, but negative by Southern blotting which suggests thepresence of the sequences in this tissue at levels below the detectionthreshold for Southern blotting.

TABLE 2 Differential detection of KS330Bam, KS627Bam and KS330₂₃₄sequences in KS-involved and non-involved tissues from three patientswith AIDS-KS. KS330Bam KS627Bam KS330₂₃₄ Patient A KS, skin + + + nlskin + + + nl muscle + + + Patient B KS, skin + + + nl muscle − − − nlbrain − − − Patient C KS, stomach + + + nl stomach − − + adjacent to KSnl muscle − − − nl brain − − − nl colon − − − nl heart − − − nl hilarlymph − − − nodes

Experiment 4 Subcloning and Sequencing of KSHV

KS330Bam and KS627Bam are genomic fragments of a novel infectious agentassociated with AIDS-KS. A genomic library from a KS lesion was made anda phage clone with a 20 kb insert containing the KS330Bam sequence wasidentified. The 20 kb clone digested with PvuII (which cuts in themiddle of the KS330Bam sequence) produced 1.1 kb and 3 kb fragments thathybridized to KS330Bam. The 1.1 kb subcloned insert and ˜900 bp from the3 kb subcloned insert resulting in 9404 bp of contiguous sequence wasentirely sequenced. This sequence contains partial and complete openreading frames homologous to regions in gamma herpesviruses.

The KS330Bam sequence is an internal portion of a 918 bp ORF with 55-56%nucleotide identity to the ORF26 and BDLF1 genes of HSVSA and EBVrespectively (SEQ IS NO:46 and 47, respectively). The EBV and HSVSAtranslated amino acid sequences for these ORFs demonstrate extensivehomology with the amino acid sequence encoded by the KS-associated 918bp ORF (FIG. 6). In HSVSA, the VP23 protein is a late structural proteininvolved in capsid construction. Reverse transcriptase (RT)-PCR of mRNAfrom a KS lesion is positive for transcribed KS330Bam 15 mRNA and thatindicates that this ORF is transcribed in KS lesions. Additionalevidence for homology between the KS agent and herpesviruses comes froma comparison of the genomic organization of other potential ORFs on the9404 bp sequence (FIG. 3A) The 5′ terminus of the sequence is composednucleotides having 66-67% nucleotide identity and 68-71% amino acididentity to corresponding regions of the major capsid protein (MCP) ORFsfor both EBV and HSVSA. This putative MCP ORF of the KS agent liesimmediately 5′ to the BDLF1/ORF26 homolog which is a conservedorientation among herpesvirus subfamilies for these two genes. At the 3end of this sequence, the reading frame has strong amino acid andnucleotide homology to HSVSA ORF 27. Thus, KS-associated DNA sequencesat four loci in two separate regions with homologies to gammaherpesviral genomes have been identified.

In addition to fragments obtained from Pvu II digest of the 21 Kb phageinsert described above, fragments obtained from a BamHI/NotI digest werealso subcloned into pBluescript (Stratagene, La Jolla, Calif.). Thetermini of these subcloned fragments were sequenced and were also foundto be homologous to nucleic acid sequence EBV and HSVSA genes. Thesehomologs have been used to develop a preliminary map of subclonedfragments (FIG. 9). Thus, sequencing has revealed that the KS agentmaintains co-linear homology to gamma herpesviruses over the length ofthe 21 Kb phage insert.

Experiment 5 Determination of the Phylogeny of KSHV

Regions flanking KS330Bam were sequenced and characterized bydirectional walking. This was performed by the following strategy: 1) KSgenomic libraries were made and screened using the KS330Bam fragment asa hybridization probe, 2) DNA inserts from phage clones positive for theKS330Bam probe were isolated and digested with suitable restrictionenzyme(s), 3) the digested fragments were subcloned into pBluescript(Stratagene, La Jolla, Calif.), and 4) the subclones were sequenced.Using this strategy, the major capsid protein (MCP) ORF homolog was thefirst important gene locus identified. Using sequenced unique 3′ and 5′end-fragments from positive phage clones as probes, and following thestrategy above a KS genomic library are screened by standard methods foradditional contiguous sequences.

For sequencing purposes, restriction fragments are subcloned intophagemid pBluescript KS+, pBluescript KS−, pBS+, or pBS− (Stratagene) orinto plasmid pUC18 or pUC19. Recombinant DNA was purified through CsCldensity gradients or by anion-exchange chromatography (Qiagen).

Nucleotide sequenced by standard screening methods of cloned fragmentsof KSHV were done by direct sequencing of double-stranded DNA usingoligonucleotide primers synthesized commercially to “walk” along thefragments by the dideoxy-nucleotide chain termination method. Junctionsbetween clones are confirmed by sequencing overlapping clones.

Targeted homologous genes in regions flanking KS330Bam include, but arenot limited to: 11-10 homolog, thymidine kinase (TK), g85, g35, gH,capsid proteins and MCP. TK is an early protein of the herpesvirusesfunctionally linked to DNA replication and a target enzyme foranti-herpesviral nucleosides. TK phosphorylates acyclic nucleosides suchas acyclovir which in turn inhibit viral DNA polymerase chain extension.Determining the sequence of this gene will aid in the prediction ofchemotherapeutic agents useful against KSHV. TK is encoded by the EBVBXLF1 ORF located ˜9700 bp rightward of BDLF1 and by the HSVSA ORF21-9200 bp rightward of the ORF 26. A subcloned fragment of KS5 wasidentified with strong homology to the EBV and HSVSA TK open readingframes.

g85 is a late glycoprotein involved in membrane fusion homologous to gHin HSV1. In EBV, this protein is encoded by BLXF2 ORF located -7600 bprightward of BDLF1, and in HSVSA it is encoded by ORF 22 located ˜7100bp rightward of ORF26.

g35 is a late EBV glycoprotein found in virion and plasma membrane. Itis encoded by BDLF3 ORF which is 1300 bp leftward of BDLF1 in EBV. Thereis no BDLF3 homolog in HSVSA. A subcloned fragment has already beenidentified with strong homology to the EBV gp35 open reading frame.

Major capsid protein (MCP) is a conserved 150 KDa protein which is themajor component of herpesvirus capsid. Antibodies are generated againstthe MCP during natural infection with most herpesviruses. The terminal1026 bp of this major capsid gene homolog in KSHV have been sequenced.

Targeted homologous genes/loci in regions flanking KS627Bam include, butare not limited to terminal reiterated repeats, LMPI, EBERs and Ori P.Terminal reiterated sequences are present in all herpesviruses. In EBV,tandomly reiterated 0.5 Kb long terminal repeats flank the ends of thelinear genome and become joined in the circular form. The terminalrepeat region is immediately adjacent to BNRF1 in EBV and ORF 75 inHSVSA. Since the number of terminal repeats varies between viralstrains, identification of terminal repeat regions may allow typing andclonality studies of KSHV in KS legions. Sequencing through the terminalrepeat region may determine whether this virus is integrated into humangenome in KS.

LMPI is an latent protein important in the transforming effects of EBVin Burkitt's lymphoma. This gene is encoded by the EBV BNRF1 ORF located˜2000 bp rightward of tegument protein ORF BNRF1 in the circularizedgenome. There is no LMP1 homolog in HSVSA.

EBERs are the most abundant RNA in latently EBV infected cells and Ori-Pis the origin of replication for latent EBV genome. This region islocated between ˜4000-9000 bp leftward of the BNRF1 ORF in EBV; thereare no corresponding regions in HSVSA.

The data indicates that the KS agent is a new human herpesvirus relatedto gamma herpesviruses EBV and HSVSA. The results are not due tocontamination or to incidental co-infection with a known herpesvirussince the sequences are distinct from all sequenced herpesviral genomes(including EBV, CMV, HHV6 and HSVSA) and are associated specificallywith KS in three separate comparative studies. Furthermore, PCR testingof KS DNA with primers specific for EBV-1 and EBV-2 failed todemonstrate these viral genomes in these tissues. Although KSHV ishomologous to EBV regions, the sequence does not match any other knownsequence and thus provides evidence for a new viral genome, related tobut distinct from known members of the herpesvirus family.

Experiment 6 Serological Studies Indirect Immunofluorescence Assay (IFA)

Virus-containing cells are coated to a microscope slide. The slides aretreated with organic fixatives, dried and then incubated with patientsera. Antibodies in the sera bind to the cells, and then excessnonspecific antibodies are washed off. An antihuman immunoglobulinlinked to a fluorochrome, such as fluorescein, is then incubated withthe slides, and then excess fluorescent immunoglobulin is washed off.The slides are then examined under a microscope and if the cellsfluoresce, then this indicates that the sera contains antibodiesdirected against the antigens present in the cells, such as the virus.

An indirect immunofluorescence assay (IFA) was performed on the BodyCavity-Based Lymphoma cell line (BCBL-1), which is a naturallytransformed EBV infected (nonproducing) B cell line, using 4 KS patientsera and 4 control sera (from AIDS patients without KS). Initially, bothsets of sera showed similar levels of antibody binding. To removenonspecific antibodies directed against EBV and lymphocyte antigens,sera at 1:25 dilution were pre-adsorbed using 3×10⁶ 1%paraformaldehyde-fixed Raji cells per ml of sera. BCBL1 cells were fixedwith ethanol/acetone, incubated with dilutions of patient sera, washedand incubated with fluorescein-conjugated goat anti-human IgG. Indirectimmunofluorescent staining was determined.

Table 3 shows that unabsorbed case and control sera have similarend-point dilution indirect immunofluorescence assay (IFA) titersagainst the BCBL1 cell line. After Raji adsorption, case sera havefour-fold higher IFA titers against BCBL1 cells than control sera.Results indicated that pre-adsorption against paraformaldehyde-fixedRaji cells reduces fluorescent antibody binding in control sera but donot eliminate antibody binding to KS case sera. These results indicatethat subjects with KS have specific antibodies directed against the KSagent that can be detected in serological assays such as IFA, Westernblot and Enzyme immunoassays (Table 3).

TABLE 3 Indirect immunofluorescence end-point titers for KS case andnon-KS control sera against the BCBL-1 cell line Sera No. Status*Pre-adsorption Post-adsorption** 1 KS ≧1:400  ≧1:400  2 KS 1:100  1:1003 KS 1:200  1:100 4 KS ≧1:400   1:200 5 Control ≧1:400  1:50 6 Control1:50  1:50 7 Control 1:100 1:50 8 Control 1:200 1:50 Legend Table 3: *KS= autopsy-confirmed male, AIDS patient Control = autopsy-confirmedfemale, AIDS patient, no KS **Adsorbed against RAJI cells treated with1% paraformaldehyde

Immunoblotting (“Western Blot”)

Virus-containing cells or purified virus (or a portion of the virus,such as a fusion protein) is electrophoresed on a polyacrylamide gel toseparate the protein antigens by molecular weight. The proteins areblotted onto a nitrocellulose or nylon membrane, then the membrane isincubated in patient sera. Antibodies directed against specific antigensare developed by incubating with a anti-human immunoglobulin attached toa reporter enzyme, such as a peroxidase. After developing the membrane,each antigen reacting against antibodies in patient sera shows up as aband on the membrane at the corresponding molecular weight region.

Enzyme Immunoassay (“EIA or ELISA”)

Virus-containing cells or purified virus (or a portion of the virus,such as a fusion protein) is coated to the bottom of a 96-well plate byvarious means (generally incubating in alkaline carbonate buffer). Theplates are washed, then the wells are incubated with patient sera.Antibodies in the sera directed against specific antigens stick on theplate. The wells are washed again to remove nonspecific antibody, thenthey are incubated with a antihuman immunoglobulin attached to areporter enzyme, such as a peroxidase. The plate is washed again toremove nonspecific antibody and then developed. Wells containing antigenthat is specifically recognized by antibodies in the patients serachange color and can be detected by an ELISA plate reader (aspectrophotomer).

All three of these methods can be made more specific by pre-incubatingpatient sera with uninfected cells to adsorb out cross-reactingantibodies against the cells or against other viruses that may bepresent in the cell line, such as EBV. Cross-reacting antibodies canpotentially give a falsely positive test result (i.e. the patient isactually not infected with the virus but has a positive test resultbecause of cross-reacting antibodies directed against cell antigens inthe preparation). The importance of the infection experiments with Rajiis that if Raji cells, or another well-defined cell line, can beinfected, then the patient's sera can be pre-adsorbed against theuninfected parental cell line and then tested in one of the assays. Theonly antibodies left in the sera after pre-adsorption that bind toantigens in the preparation should be directed against the virus.

Experiment 7

BCBL 1, from lymphomatous tissues belonging to a rare infiltrating,anaplastic body cavity lymphoma occurring in AIDS patients has beenplaced in continuous cell culture and shown to be continuously infectedwith the KS agent. This cell line is also naturally infected withEpstein-Barr Virus (EBV). The BCBL cell line was used as an antigensubstrate to detect specific KS antibodies in persons infected with theputative virus by Western-blotting. Three lymphoid B cell lines wereused as controls. These included the EBV genome positive cell line P3H3,the EBV genome defective cell line Raji and the EBV genome negative cellline Bjab.

Cells from late-log phase culture were washed 3 time with PBS bycentrifugation at 500 g for 10 min. and suspended in sample buffercontaining 50 mM Tris-HCl pH 6.8, 2% SDS (w/v), 15% glycerol (v/v), 5%β-mercaptoethanol (v/v) and 0.001% bromophenol (w/v) with proteaseinhibitor, 100 μM phenylmethylsulfonyl fluoride (PMSF). The sample wasboiled at 100° C. for 5 min and centrifuged at 14,000 g for 10 min. Theproteins in the supernatant was then fractionated by sodium, dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducingconditions with a separation gel of 15% and a stacking gel of 5% (3).Prestained protein standards were included: myosin, 200 kDa;β-galactosidase, 118 kDA; BSA, 78 kDa; ovalbumin, 47.1 kDa; carbonicanhydrase, 31.4 kDa; soybean trypsin inhibitor, 25.5 kDa, lysozyme, 18.8kDa and aprotinin, 8.3 kDa (Bio-Rad). Immunoblotting experiments wereperformed according to the method of Towbin et al. (4). Briefly, theproteins were electrophoretically transferred to Hybon-C extra membranes(Pharmacia) at 24 V for 70 min. The membranes were then dried at 37° C.for 30 min, saturated with 5% skim milk in Tris-buffered saline, pH 7.4(TBS) containing 50 mM Tris-HCl and 200 mM NaCl, at room temperature for1 h. The membranes were subsequently incubated with human sera atdilution 1:200 in 1% skim milk overnight at room temperature, washed 3times with a solution containing TBS, 0.2% Triton X-100 and 0.05% skimmilk and then 2 times with TBS. The membranes were then incubated for 2h at room temperature with alkaline phosphatase conjugated goatanti-mouse IgG+IgM+IgA (Sigma) diluted at 1:5000 in 1% skim milk. Afterrepeating the washing, the membranes were stained with nitrobluetetranolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidinesalt (Gibco BRL).

Two bands of approximately 226 kDa and 234 kDa were identified to bespecifically present on the Western-blot of BCBL cell lysate in 5 serafrom AIDS gay man patients infected with KS. These 2 bands were absentfrom the lysates of P3H3, Raji and Bjab cell lysates. 5 sera from AIDSgay man patients without KS and 2 sera from AIDS woman patients withoutKS as well as 1 sera from nasopharyncel carcinoma patient were not ableto detect these 2 bands in BCBL 1, P3H3, Raji and Bjab cell lysates. Ina blinded experiment, using the 226 kDa and 234 kDa markers, 15 out of16 sera from KS patients were correctly identified. In total, the 226kDa and 234 kDa markers were detected in 20 out of 21 sera from KSpatients.

The antigen is enriched in the nuclei fraction of BCBL1. Enrichedantigen with low background can be obtained by preparing nucleic fromBCBC as the starting antigen preparation using standard, widelyavailable protocols. For example, 500-750 ml of BCBL at 5×10⁵ cells/mlcan be pelleted at low speed. The pellet is placed in 10 mM NaCl, 10 mMThis pH 7.8, 1.5 mM MgCl₂ (equi volume)+1.0% NP-40 on ice for 20 min tolyse cells. The lysate is then spun at 1500 rpm for 10 min. to pelletnucleic. The pellet is used as the starting fraction for the antigenpreparation for the Western blot. This will reduce cross-reactivecytoplasmic antigens.

Experiment 8 Transmission Studies Co-Infection Experiments

BCBL1 cells were co-cultivated with Raji cell lines separated by a 0.45μtissue filter insert. Approximately, 1-2×10⁶ BCBL1 and 2×10⁶ Raji cellswere co-cultivated for 2-20 days in supplemented RPMI alone, in 10 μg/ml5′-bromodeoxyuridine (BUdR) and 0.6 μg/ml 5′-fluorodeoxyuridine or 20ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA). After 2, 8, 12 or 20days co-cultivation, Raji cells were removed, washed and placed insupplemented RPMI 1640 media. A Raji culture co-cultivated with BCBL1 in20 ng/ml TPA for 2 days survived and has been kept in continuoussuspension culture for >10 weeks. This cell line, designated RCC1 (RajiCo-Culture, No. 1) remains PCR positive for the KS330₂₃₄ sequence aftermultiple passages. This cell line is identical to its parental Raji cellline by flow cytometry using EMA, B1, B4 and BerH2 lymphocyte-flowcytometry (approximately 2%). RCC1 periodically undergo rapid cytolysissuggestive of lytic reproduction of the agent. Thus, RCC1 is a Raji cellline newly infected with KSHV.

The results indicate the presence of a new human virus, specifically aherpesvirus in KS lesions. The high degree of association between thisagent and AIDS-KS (>90%), and the low prevalence of the agent in non-KStissues from immunocompromised AIDS patients, indicates that this agenthas a causal role in AIDS-KS [47, 68].

Experiment 10 Isolation of KSHV

Crude virus preparations are made from either the supernatant or lowspeed pelleted cell fraction of BCBL1 cultures. Approximately 650 ml ormore of log phase cells should be used (>5×10⁶ cells/ml).

For bonding whole virion from supernatant, the cell free supernatant isspun at 10,000 rpm in a GSA rotor for 10 min to remove debris. PEG-8000is added to 7%, dissolved and placed on ice for >2.5 hours. ThePEG-supernatant is then spun at 10,000×g for 30 min. supernatant ispoured off and the pellet is dried and scraped together from thecentrifuge bottles. The pellet is then resuspended in a small volume(1-2 ml) of virus buffer (VB, 0.1 M NaCl, 0.01 M Tris, pH 7.5). Thisprocedure will precipitate both naked genome and whole virion. Thevirion are then isolated by centrifugation at 25,000 rpm in a 10-50%sucrose gradient made with VB. One ml fractions of the gradient are thenobtained by standard techniques (e.g. using a fractionator) and eachfraction is then tested by dot blotting using specific hybridizingprimer sequences to determine the gradient fraction containing thepurified virus (preparation of the fraction maybe needed in order todetect the presence of the virus, such as standard DNA extraction).

To obtain the episomal DNA from the virus, the pellet of cells is washedand pelleted in PBS, then lysed using hypotonic shock and/or repeatedcycles of freezing and thawing in a small volume (<3 ml).

Nuclei and other cytoplasmic debris are removed by centrifugation at10,000 g for 10 min, filtration through a 0.45 m filter and then repeatcentrifugation at 10,000 g for 10 min. This crude preparation containsviral genome and soluble cell components. The genome preparation canthen be gently chloroform-phenol extracted to remove associated proteinsor can be placed in neutral DNA buffer (1 M NaCl, 50 mM Tris, 10 mMEDTA, pH 7.2-7.6) with 2% sodium dodecylsulfate (SDS) and 1% sarcosyl.The genome is then banded by centrifugation through 10-30% sucrosegradient in neutral DNA buffer containing 0.15% sarcosyl at 20,000 rpmin a SW 27.1 rotor for 12 hours (for 40,000 rpm for 2-3 hours in an SW41rotor). The band is detected as described above.

An example of the method for isolating KSHV genome from KSHV infectedcell cultures (97 and 98). Approximately 800 ml of BCBL1 cells arepelleted, washed with saline, and pelleted by low speed centrifugation.The cell pellet is lysed with an equal volume of RSB (10 mM NaCl, 10 mMTris-HCl, 1.5 mM MgCl2, pH 7.8) with 1% NP-40 on ice for 10 minutes. Thelysate is centrifuged at 900×g for 10 minutes to pellet nuclei. Thisstep is repeated. To the supernatant is added 0.4% sodium dodecylsulfateand EDTA to a final concentration of 10 mM. The supernatant is loaded ona 10-30% sucrose gradient in 1.0 M NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH7.5. The gradients are centrifuged at 20,000 rpm on a SW 27.1 rotor for12 hours. In FIG. 11, 0.5 ml aliquots of the gradient have beenfractionated (fractions 1-62) with the 30% gradient fraction being atfraction No. 1 and the 10% gradient fraction being at fraction No. 62.Each fraction has been dot hybridized to a nitrocellulose membrane andthen a ³²P-labeled KSHV DNA fragment, KS631Bam has been hybridized tothe membrane using standard techniques. FIG. 11 shows that the majorsolubilized fraction of the KSHV genome bands (i.e. is isolated) infractions 42 through 48 of the gradient with a high concentration of thegenome being present in fraction 44. A second band of solubilized KSHVDNA occurs in fractions 26 through 32.

Experiment 11 Purification of KSHV

DNA is extracted using standard techniques from the RCC-1 or RCC-1_(2F5)cell line [27, 49, 66]. The DNA is tested for the presence of the KSHVby Southern blotting and PCR using the specific probes as describedhereinafter. Fresh lymphoma tissue containing viable infected cells issimultaneously filtered to form a single cell suspension by standardtechniques [49, 66]. The cells are separated by standard Ficoll-Plaquecentrifugation and lymphocyte layer is removed. The lymphocytes are thenplaced at >1×10⁶ cells/ml into standard lymphocyte tissue culturemedium, such as RMP 1640 supplemented with 10% fetal calf serum.Immortalized lymphocytes containing the KSHV virus are indefinitelygrown in the culture media while nonimmortilized cells die during courseof prolonged cultivation.

Further, the virus may be propagated in a new cell line by removingmedia supernatant containing the virus from a continuously infected cellline at a concentration of >1×10⁶ cells/ml. The media is centrifuged at2000×g for 10 minutes and filtered through a 0.45μ filter to removecells. The media is applied in a 1:1 volume with cells growing at >1×10⁶cells/ml for 48 hours. The cells are washed and pelleted and placed infresh culture medium, and tested after 14 days of growth.

The herpesvirus may be isolated from the cell DNA in the followingmanner. An infected cell line, which can be lysed using standard methodssuch as hyposmotic shocking and Dounce homogenization, is first pelletedat 2000×g for 10 minutes, the supernatant is removed and centrifugedagain at 10,000×g for 15 minutes to remove nuclei and organelles. Thesupernatant is filtered through a 0.45μ filter and centrifuged again at100,000×g for 1 hour to pellet the virus. The virus can then be washedand centrifuged again at 100,000×g for 1 hour.

REFERENCES

-   1. Ablashi, D. V. et al. Virology 184:545-552.-   2. Albrecht, J. C., et al. (1992) J. Virol. 66:5047.-   3. Altshul, S. F. et al. (1990) J. Molec. Biol. 215:403.-   4. Analytical Biochemistry (1984) 238:267-284.-   5. Andrei, et al. (1992) Eur. J. Clin. Microbiol. Infect. Dis.    11(2):143-51.-   6. Archibald, C. P., at al. (1992) Epidemiol. 3:203.-   7. Asada, H., et al (1989) J. Clin. Microbiol. 27(10):2204.-   8. Ausubel, F., et al. (1987) Current Protocols in Molecular    Biology, New York.-   9. Baer, R. J., et al. (1984) Nature 310:207.-   10. Bagasra, et al. (1992) J. New England Journal of Medicine    326(21):1385-1391.-   11. Balzarini, et al. (1990) Mol. Pharm. 37, 402-7.-   12. Basic and Clinical Immunology 7th Edition D. Stites and A. Terr    ed.-   13. Beral, V., et al. (1990) Lancet 335:123.-   14. Beral, V., at al. (1991) Brit. Med. J. 302:624.-   15. Beral, V., et al. (1992) Lancet 339:632.-   16. Bendsöe, N., et al. (1990) Eur. J. Cancer 26:699.-   17. Biggar, R. J., et al. (1994) Am. J. Epidemiol. 139:362.-   18. Bovenzi, P., et al. (1993) Lancet 341:1288.-   19. Beaucage and Carruthers (1981) Tetrahedron Lett. 22:1859-1862.-   20. Braitman, et al. (1991) Antimicrob. Agents and Chemotherapy    35(7):1464-8.-   21. Burns and Sanford, (1990) J. Infect. Dis. 162(3):634-7.-   22. De Clercq, (1993) Antimicrobial Chemotherapy 32, Suppl. A,    121-132.-   23. Drew, W. L., et al. (1982) Lancet ii:125.-   24. Falk, et al. (1991) Nature 351:290.-   25. Gaidano, G., et al. (1991) Proc. Natl. Acad. Sci. USA 88:5413.-   26. Gershon, A. A., (1992) J. Inf. Des. 166(Suppl):563.-   27. Glick, J. L., (1980) Fundamentals of Human Lymphoid Culture,    Marcel Dokker, New York.-   28. Gorbach, S. L., et al. (1992) Infectious Disease Ch.    35:289, W. B. Saunders, Philadelphia, Pa.-   29. Greenspan, et al. (1990) J. Acquir. Immune Defic. Syndr. 3    (6):571.-   30. Hardy, I., et al. (1990) Inf. Dis. Clin. N. Amer. 4(1):159.-   31. Hardy, I., et al. (1991) New Engl. J. Med. 325 (22):1545.-   31A. Harel-Bellan, A., et al. (1988) Exp. Med. 168:2309-2318-   32. Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold    Spring Harbor Publication, New York.-   33. Haverkos, H. W., et al. (1985) Sexually Transm. Dis. 12:203.-   34. Helene, C. and Toulme, J. (1990) Biochim. Biophys. Acta.    1049:99-125.-   35. Heniford, et al. (1993) Nucleic Acids Research 21(14):3159-3166.-   36. Higashi, K., et al. (1989) J. Clin. Micro. 27(10):2204.-   37. Holmberg, S. D., et al. (1990) Cancer Detection and Prevention    14:331.-   38. Holliday, J., and Williams, M. V., (1992) Antimicrob, Agents    Chemother. 36(9):1935.-   39. Hoogenboom, H. R., et al. (1991) Nuc. Acids Res. 19:4133.-   40. Hunt, et al. (1991) Eur. J. Immunol. 21:2963-2970.-   41. Hybridization of Nucleic Acids Immobilized on Solid Supports    Meinkoth, J. and Wahl, G.-   42. Hybridization with Nucleic Acid Probes pp. 495-524, (1993)    Elsevier, Amsterdam.-   43. Ickes, et al. (1994) Antiviral Research 23, Seventh    International Conf. on Antiviral Research, Abstract No. 122, Supp.    1.-   44. Jahan, N., et al. (1989) AIDS Research and Human Retroviruses    5:225.-   45. Jardetzkey, et al. (1991) Nature 353:326.-   46. Johnston, G. S., et al. (1990) Cancer Detection and Prevention    14:337.-   47. Jung, J. U., et al. (1991) Proc. Natl. Acad. Sci. USA 88:7051.-   48. Kikuta, et al. (1989) Lancet Oct. 7:861.-   49. Knowles, D. M., et al. (1989) Blood 73:792-798.-   50. Kohler and Milstein, (1976) Eur. J. Immunol 6:511-519.-   51. Kucera, et al. (1993) AIDS Res. Human Retroviruses 9:307-314.-   52. Laboratory Techniques in Biochemistry and Molecular    Biology (1978) North Holland Publishing Company, New York.-   53. Lasky, L. A., (1990) J. Med. Virol. 31(1):59.-   54. Levin, M. J., et al. (1992) J. Inf. Dis. 166(2):253.-   55. Lifson, A. R., et al., (1990) Am. J. Epidemiol. 131:221.-   56. Lin, et al. (1991) Antimicrob Agents Chemother 35(11):2440-3.-   57. Lin, J. C., et al. (1993) Blood 81:3372.-   58. Lisitsyn, N., et al. (1993) Science 259:946.-   59. Lo, S-C., et al. (1992) Internat. J. Systematic Bacterial.    42:357.-   60. Marks, J. D., et al. (1991) J. Mol. Biol. 222:581-597.-   61. Marloes, et al. (1991) Eur. J. Immunol. 21:2963-2970.-   62. Matteucci, et al. (1981) Am. Chem. Soc. 103:3185.-   63. Maxam, A. M. and Gilbert, W. Methods in Enzymology (1980)    Grossman, L. and Moldave, D. eds., Academic Press, New York,    65:499-560.-   64. McCafferty, J., et al. (1990) Nature 348:552.-   65. Means and Feeney, (1990) Bioconjugate Chem. A recent review of    protein modification techniques, 1:2-12.-   66. Metcalf, D. (1984) Clonal Culture of Hematopoeitic Cells:    Techniques and Applications, Elvier, New York.-   67. Methods in Enzymology Vol. 152, (1987) Berger, S. and Kimmel, A.    ed., Academic Press, New York-   68. Miller, G., Virology (1990) B. N. Fields, D. M. Knipe eds.,    Raven Press, New York, 2:1921.-   69. Needham-VanDevanter, D. R., et al., (1984) Nucleic Acids Res.    12:6159-6168.-   70. Needleman and Wunsch, (1970) J. Mol. Biol. 48:443.-   71. Neuvo, et al. (1993) American Journal of Surgical Pathology    17(7), 683-690.-   72. Nucleic Acid Hybridization: A Practical Approach (1985) Ed.    Hames, B. D. and Higgins, S. J., IRL Press.-   73. Oren and Soble, (1991) Clinical Infectious Diseases 14:741-6.-   74. PCR Protocols: A Guide to Methods and Applications. (1990)    Innis, M., Gelfand D., Sninsky, J. and White, T., eds., Academic    Press, San Diego.-   75. Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. (USA) 85:2444.-   75A. Pearson, J. D., and Regnier, F. E., (1983) J. Chrom.    255:137-14976.-   76. Pellici, P. G., et al. (1985) J. Exp. Med. 162:1015.-   77. Peterman, T. A., et al. (1991) Cancer Surveys Imperial Cancer    Research Fund, London, 10:23-37.-   78. Roizman, B. (1991) Rev. Inf. Disease 13 Suppl. 11:S892.-   79. Rötzschke and Falk, (1991) Immunol. Today 12:447.-   80. Safai, B., et al. (1980) Cancer 45:1472.-   81. Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual    (2nd ed.), Cold Spring Harbor Laboratory, Vols. 1-3.-   82. Saunders, et al. (1990) J. Acquir. Immune Defic. Syndr. 3    (6):571.-   83. Schecter, M. T., et al. (1991) Am. J. Epidemiol. 134:485.-   84. Scopes, R., (1982) Protein Purification: Principles and Practice    Springer-Verlag, New York.-   85. Siddiqui, A., et al., (1983) Proc. Natl. Acad. Sci. USA 80:4861.-   86. Skinner, G. R., et al. (1991) Comp. Immuno. Microbiol. Inf. Dis.    14(2):13.-   87. Skinner, G. R., et al. (1992) Med. Microbiol. Immunol.    180(6):305. Smith and Waterman (1981) Adv. Appl. Math. 2:482.-   88. Snoeck, et al. (1992) Eur. J. Clin. Micro. Infect. Dis.    11(12):1144-55.-   89. Stals, et al. (1993) Antimicrobial Agents Chemother.    37(2):218-23.-   90. van den Berg, F. et al. (1989) J. Clin. Pathol. 42:128.-   91. Vogel, J., et al. (1988) Nature 335:606.-   92. Wang, R. H.-Y., et al. (1993) Clin. Infect. Dis. 17:724.-   93. Wickstrom, E. L., et al. (1988)PNAS (USA) 85:1028-1032.-   94. Winkelmann, et al. (1988) Drug Res. 38, 1545-48.-   95. Winkler, et al. (1990) Antiviral Research 14:61-74.-   96. Yamandaka, et al. (1991) Mol. Pharmacol. 40(3):446.-   97. Pellicer, A. et al. (1978) Cell 14:133-141.-   98. Gibson, W. and Roizmann B. (1972) J. Virol. 10:1044-52.

EXPERIMENTAL DETAILS SECTION II

Sequencing Studies: A lambda phage (KS5) from a KS lesion genomiclibrary identified by positive hybridization with KS330Bam was digestedwith BamHI and Not I (Boehringer-Mannheim, Indianapolis Ind.); fivefragments were gel isolated and subcloned intra Bluescript II KS(Stratagene, La Jolla Calif.). The entire sequence was determined bybidirectional sequencing at a seven fold average redundancy by primerwalking and nested deletions.

DNA sequence data were compiled and aligned using ALIGN (IBI-Kodak,Rochester N.Y.) and analyzed using the Wisconsin Sequence AnalysisPackage Version 8-UNIX (Genetics Computer Group, Madison Wis.) and theGRAIL Sequence Analysis, Gene Assembly and Sequence Comparison System v.1.2 (Informatics Group, Oak Ridge Tenn.). Protein site motifs wereidentified using Motif (Genetics Computer Group, Madison Wis.).

Sources of Herpesvirus Gene Sequence Comparisons: Complete genomicsequences of three gammaherpesviruses were available: Epstein-Barr virus(EBV), a herpesvirus of humans [4]; herpesvirus saimiri (HVS), aherpesvirus of the New World monkey Saimiri sciureus [1]; and equineherpesvirus 2 (EHV2 [49]). Additional thymidine kinase gene sequenceswere obtained for alcelaphine herpesvirus 1 (AHV1 [22]) and bovineherpesvirus 4 (BHV4 [31]). Sequences for the major capsid protein genesof human herpesvirus 6B and human herpesvirus 7 (HHV7) were from Mukaiat al. [34]. The sources of all other sequences used are listedpreviously in McGeoch and Cook [31] and McGeoch et al. [32].

Phylogenetic Inference: Predicted amino acid sequences used for treeconstruction were based on previous experience with herpesviralphylogenetic analyses [31]. Alignments of homologous sets of amino acidsequences were made with the AMPS [5] and Pileup [16] programs. Regionsof alignments that showed extreme divergence with marked lengthheterogeneity, typically terminal sections, were excised. Generally,positions in alignments that contained inserted gaps in one or moresequences were removed before use for tree construction. Phylogeneticinference programs were from the Phylip set, version 3.5c [14] and fromthe GCG set [16]. Trees were built with the maximum parsimony (MP),neighbor joining (NJ) methods. For the NJ method, which utilizesestimates of pairwise distances between sequences, distances wereestimated as mean numbers of substitution events per site with Protdistusing the PAM 250 substitution probability matrix of Schwartz & Dayhoff[46]. Bootstrap analysis [15] was carried out for MP and NJ trees, with100 sub-replicates of each alignment, and consensus trees obtained withthe program Consense. In addition the program Protml was used to infertrees by the maximum likelihood (ML) method. Protml was obtained form J.Adachi, Department of Statistical Science, The Graduate University forAdvanced Study, Tokyo 106, Japan. Because of computational constraints,Protml was used only with the 4-species CS1 alignment.

Clamped Homogeneous Electric Field (CHEF) Gel Electrophoresis: Agaroseplugs were prepared by resuspending BCBL-1 cells in 1% LMP agarose(Biorad, Hercules Calif.) and 0.9% NaCl at 42° C. to a finalconcentration of 2.5×10⁷ cells/ml. Solidified agarose plugs weretransferred into lysis buffer (0.5M EDTA pH 8.0, 1% sarcosyl, proteinaseK at 1 mg/ml final concentration) and incubated for 24 hours.Approximately 10⁷ BCBL-1 cells were loaded in each lane. Gels were runat a gradient of 6.0 V/cm with a run time of 28 h 28 min. on a CHEFMapper XA pulsed field gel electrophoresis apparatus (Biorad, HerculesCalif.), Southern blotted and hybridized to KS627Bam, KS330Bam and anEBV terminal repeat sequence [40].

TPA Induction of Genome Replication: Late log phase BCBL-1 cells (5×10⁵cells per ml) were incubated with varying amounts of12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma Chemical Co., St. LouisMo.) for 48 h, cells were then harvested and washed withphosphate-buffered saline (PBS) and DNA was isolated bychloroform-phenol extraction. DNA concentrations were determined by UVabsorbance; 5 μg of whole cell DNA was quantitatively dot blothybridized in triplicate (Manifold I, Schleicher and Schuell, KeeneN.H.). KS631Bam, EBV terminal repeat and beta-actin sequences wererandom-primer labeled with ³²P [13]. Specific hybridization wasquantitated on a Molecular Dynamics Phosphorlmager 425E.

Cell Cultures and Transmission Studies: Cells were maintained at 5×10⁵cells per ml in RPMI 1640 with 20% fetal calf serum (FCS, Gibco-BRL,Gaithersburg Md.) and periodically examined for continued KSHV infectionby PCR and dot hybridization. The T cell line Molt-3 (a gift from Dr.Jodi Black, Centers for Disease Control and Prevention), Raji cells(American Type Culture Collection, Rockville Md.) and RCC-1 cells werecultured in RPMI 1640 with 10% FCS. Owl monkey kidney cells (AmericanType Culture Collection, Rockville Md.) were cultured in MEM with 10%FCS and 1% nonessential amino acids (Gibco-BRL, Gaithersburg Md.).

To produce the RCC-1 cell line, 2×10⁶ Raji cells were cultivated with1.4×10⁶ BCBL-1 cells in the presence of 20 ng/ml TPA for 2 days inchambers separated by Falcon 0.45 μg filter tissue culture inserts toprevent contamination of Raji with BCBL-1. Demonstration that RCC-1 wasnot contaminated with BCBL-1 was obtained by PCR typing of HLA-DRalleles [27] (Raji and RCC-1: DRβ1*0310, DRβ3*02; BCBL-1: DRβ104,*07,Drβ4*01) and confirmed by flow cytometry to determine the presence(Raji, RCC1) or absence (BCBL-1) of EMA membrane antigen. Clonalsublines of RCC-1 were obtained by dilution in 96 well plates to 0.1cells/well in RPMI 1640, 20% FCS and 30% T-STIM culture supplement(Collaborative Biomedical Products, Bedford Mass.). Subcultures wereexamined to ensure that each was derived from a single cluster ofgrowing cells.

In situ hybridization was performed with a previously described 25 bpoligomer located in ORF26 which was 5′ labeled with fluorescein (Operon,Alameda Calif.) and hybridized to cytospin preparations of BCBL-1, RCC-1and Raji cells using the methods of Lungu et al. [29]. Slides were bothdirectly visualized by UV microscopy and by incubating slides withanti-fluorescein-alkaline phosphatase (AP)-conjugated antibody(Boehringer-Mannheim, Indianapolis Ind.), allowing immunohistochemicaldetection of bound probe. Positive control hybridization was performedusing a 26 bp TET-labeled EBV DNA polymerase gene oligomer (AppliedBiosystems, Alameda Calif.) which was visualized by UV microscopy onlyand negative control hybridization was performed using a 25 bp 5′fluorescein-labeled HSV1 α47 gene oligomer (Operon, Alameda Calif.)which was visualized in a similar manner as the KSHV ORF26 probe. Allnuclei of BCBL-1, RCC-1 and Raji appropriately stained with the EBVhybridization probe whereas no specific staining of the cells occurredafter hybridization with the HSV1 probe.

The remaining suspension cell lines used in transmission experimentswere pelleted, and resuspended in 5 ml of 0.22 or 0.45μ filtered BCBL-1tissue culture supernatant for 16 h. BCBL-1 supernatants were eitherfrom unstimulated cultures or from cultures stimulated with 20 ng/mlTPA. No difference in transmission to recipient cell lines was notedusing various filtration or stimulation conditions. Fetal cord bloodlymphocytes (FCBL) were obtained from heparinized fresh post-partumumbilical cord blood after separation on Ficoll-Paque (Pharmacia LKB,Uppsala Sweden) gradients and cultured in RPMI 1640 with 10% fetal calfserum. Adherent recipient cells were washed with sterile Hank's BufferedSalt Solution (HBSS, Gibco-BRL, Gaithersburg Md.) and overlaid with 5 mlof BCBL-1 media supernatant. After incubation with BCBL-1 mediasupernatant, cells were washed three times with sterile HBSS, andsuspended in fresh media. Cells were subsequently rewashed three timesevery other day for six days and grown for at least two weeks prior toDNA extraction and testing. PCR to detect KSHV infection was performedusing nested and unnested primers from ORF 26 and ORF 25 as previouslydescribed [10, 35].

Indirect Immunofluorescence Assay: AIDS-KS sera were obtained fromongoing cohort studies (provided by Drs. Scott Holmberg, Thomas Spiraand Harold Jaffe, Centers for Disease Control, and Prevention, and IsaacWeisfuse, New York City Department of Health).

Sera from AIDS-KS patients were drawn between 1 and 31 months afterinitial KS diagnosis, sera from intravenous drug user andhomosexual/bisexual controls were drawn after non-KS AIDS diagnosis, andsera from HIV-infected hemophiliac controls were drawn at various timesafter HIV infection. Immunofluorescence assays were performed using anequal volume mixture of goat anti-human IgG-FITC conjugate (MolecularProbes, Eugene Oreg.) and goat anti-human IgM-FITC conjugate (SigmaChemical Co., St. Louis Mo.) diluted 1:100 and serial dilutions ofpatient sera. End-point titers were read blindly and specificimmunoglobulin binding was assessed by the presence or absence of aspecular fluorescence pattern in the nuclei of the plated cells. Toadsorb cross-reacting antibodies, 20 μl serum diluted 1:10 inphosphate-buffer saline (PBS), pH 7.4, were adsorbed with 1-3×10⁷paraformaldehyde-fixed P3H3 cells for 4-10 h at 25° C. and removed bylow speed centrifugation. P3H3 were induced prior to fixation with 20ng/ml TPA for 48 h, fixed with 1% paraformaldehyde in PBS for 2 h at 4°C., and washed three times in PBS prior to adsorption.

Results Sequence Analysis of a 20.7 kb KSHV DNA Sequence:

To demonstrate that KS330Bam and KS631Bam are genomic fragments from anew and previously uncharacterized herpesvirus, a lambda phage clone(KS5) derived from an AIDS-KS genomic DNA library was identified byhybridization to the KS330Bam sequence. The KS5 insert was subclonedafter NotI/BamHI digestion into five subfragments and both strands ofeach fragment were sequenced by primer walking or nested deletion with a7-fold average redundancy. The KS5 sequence is 20,705 bp in length andhas a G+C content of 54.0%. The observed/expected CpG dinucleotide ratiois 0.92 indicating no overall CpG suppression in this region.

Open reading frame (ORF) analysis identified 15 complete ORFs withcoding regions ranging from 231 bp to 4128 bp in length, and twoincomplete ORFs at the termini of the KS5 clone which were 135 and 552by in length (FIG. 12). The coding probability of each ORF was analyzedusing GRAIL 2 and CodonPreference which identified 17 regions havingexcellent to good protein coding probabilities. Each region is within anORF encoding a homolog to a known herpesvirus gene with the exception ofone ORF located at the genome position corresponding to ORF28 inherpesvirus saimiri (HVS). Codon preference values for all of the ORFswere higher across predicted ORFs than in non-coding regions when usinga codon table composed of KS5 homologs to the conserved herpesvirusmajor capsid (MCP), glycoprotein H (gH), thymidine kinase (TK), and theputative DNA packaging protein (ORF29a/ORF29b) genes.

The translated sequence of each ORF was used to search GenBank/EMBLdatabases with BLASTX and FastA algorithms [2, 38]. All of the putativeKS5 ORFs, except one, have sequence and collinear positional homology toORFs from gamma-2 herpesviruses, especially HVS and equine herpesvirus 2(EHV2). Because of the high degree of collinearity and amino acidsequence similarity between KSHV and HVS, KSHV ORFs have been namedaccording to their HVS positional homologs (i.e. KSHV ORF25 is namedafter HVS ORF 25).

The KS5 sequence spans a region which includes three of the sevenconserved herpesvirus gene blocks (FIG. 14) [10]. ORFs present in theseblocks include genes which encode herpesvirus virion structural proteinsand enzymes involved in DNA metabolism and replication. Amino acididentities between KS5 ORFs and HVS ORFs range from 30% to 60%, with theconserved MCP ORF25 and ORF29b genes having the highest percentage aminoacid identity to homologs in other gammaherpesviruses. KSHV ORF28, whichhas no detectable sequence homology to HVS or EBV genes, has positionalhomology to HVS ORF28 and EBV BDLF3. ORF28 lies at the junction of twogene blocks (FIG. 14); these junctions tend to exhibit greater sequencedivergence than intrablock regions among herpesviral genomes [17]. TwoORFs were identified with sequence homology to the putative splicedprotein packaging genes of HVS (ORF29a/ORF29b) and herpes simplex virustype 1 (UL15). The KS330Bam sequence is located within KSHV ORF26, whoseHSV-1 counterpart, VP23, is a minor virion structural component.

For every KSHV homolog, the HVS amino acid similarity spans the entiregene product, with the exception of ORF21, the TK gene. The KSHV TKhomolog contains a proline-rich domain at its amino terminus (nt20343-19636; as 1-236) that is not conserved in other herpesvirus TKsequences, while the carboxyl terminus (nt 19637-18601; as 237-565) ishighly similar to the corresponding regions of HVS, EHV2, and bovineherpesvirus 4 (BHV4) TK. A purine binding motif with a glycine-richregion found in herpesviral TK genes, as well as other TK genes, ispresent in the KSHV TK homolog (GVMGVGKS; as 260-267).

The KS5 translated amino acid sequences were searched against thePROSITE Dictionary of Protein Sites and Patterns (Dr. Amos Bairoch,University of Geneva. Switzerland) using the computer program Motifs.Four sequence motif matches were identified among KSHV hypotheticalprotein sequences. These matches included: (i) a cytochrome c familyheme-binding motif in ORF33 (CVHCHG; as 209-214) and ORF34 (CLLCHI; as257-261), (ii) an immunoglobulin and major histocompatibility complexprotein signature in ORF25 (FICQAKH; as 1024-1030), (iii) amitochondrial energy transfer protein motif in ORF26 (PDDITRMRV; as260-268), and (iv) the purine nucleotide binding site identified inORF21. The purine binding motif is the only motif with obviousfunctional significance. A cytosine-specific methylase motif present inHVS ORF27 is not present in KSHV ORF27. This motif may play a role inthe methylation of episomal DNA in cells persistently infected with HVS[1].

Phylogenetic Analysis of KSHV: Amino acid sequences translated from theKS5 sequence were aligned with corresponding sequences from otherherpesviruses. On the basis of the level of conserved aligned residuesand the low incidence of introduced gaps, the amino acid alignments forORFs 21, 22, 23, 24, 25, 26, 29a, 29b, 31 and 34 were suitable forphylogenetic analyses.

To demonstrate the phylogenetic relationship of KSHV to otherherpesviruses, a single-gene comparison was made for ORF25 (MCP)homologs from KS5 and twelve members of Herpesviridae (FIGS. 15A-15B).The thirteen available MCP amino acid sequences are large (1376 a.a.residues for the KSHV homolog) and alignment required only a low levelof gapping; however, the overall similarity between viruses isrelatively low [33]. The MCP set gave stable trees with high bootstrapscores and assigned the KSHV homolog to the gamma-2 sublineage (genusRhadinovirus), containing HVS, EHV2 and BVH4 [20, 33, 43]. KSHV was mostclosely associated with HVS. Similar results were obtained forsingle-gene alignments of TK and UL15/ORF29 sets but with lowerbootstrap scores so that among gamma-2 herpesvirus members branchingorders for EHV2, HVS and KSHV were not resolved.

To determine the relative divergence between KSHV and othergammaherpesviruses, alignments for the nine genes listed above wereconcatenated to produce a combined gammaherpesvirus gene set (CS1)containing EBV, EHV2, HVS and KSHV amino acid sequences. The totallength of CS1 was 4247 residues after removal of positions containinggaps introduced by the alignment process in one or more of thesequences. The CS1 alignment was analyzed by the ML method, giving thetree shown in FIG. 15B and by the MP and NJ methods used with thealigned herpesvirus MCP sequences. All three methods identified KSHV andHVS as sister groups, confirming that KSHV belongs in the gamma-2sublineage with HVS as its closest known relative. It was previouslyestimated that divergence of the HVS and EHV2 lineages may have beencontemporary with divergence of the primate and ungulate host lineages[33]. The results for the CS1 set suggest that HVS and KSHV represent alineage of primate herpesviruses and, based on the distance between KSHVand HVS relative to the position of EHV2, divergence between HVS andKSHV lines is ancient.

GenomiC Studies of KSHV:

CHEF electrophoresis performed on BCBL-1 cells embedded in agarose plugsdemonstrated the presence of a nonintegrated KSHV genome as well as ahigh molecular weight species (FIGS. 16A-16B). KS631Bam (FIG. 16A) andKS33 Bam specifically hybridized to a single CHEF gel band comigratingwith 270 kilobase (kb) linear DNA standards. The majority of hybridizingDNA was present in a diffuse band at the well origin; a low intensityhigh molecular weight (HMW) band was also present immediately below theorigin (FIG. 16A. arrow). The same filter was stripped and probed withan EBV terminal repeat sequence [40] yielding a 150-160 kb band (FIG.16B) corresponding to linear EBV DNA [24]. The HMW EBV band maycorrespond to either circular or concatemeric EBV DNA [24].

The phorbol ester TPA induces replication-competent EBV to enter a lyticreplication cycle [49]. To determine if TPA induces replication of KSHVand EBV in BCBL-1 cells, these cells were incubated with varyingconcentrations of TPA for 48 h (FIG. 17). Maximum stimulation of EBVoccurred at 20 ng/ml TPA which resulted in an eight-fold increase inhybridizing EBV genome. Only a 1.3-1.4 fold increase in KSHV genomeabundance occurred after 20-80 ng/ml TPA incubation for 48 h.

Transmission Studies:

Prior to determining that the agent was likely to be a member ofHerpesviridae by sequence analysis, BCBL-1 cells were cultured with Rajicells, a nonlytic EBV transformed B cell line, in chambers separated bya 0.45μ tissue culture filter. Recipient Raji cells generallydemonstrated rapid cytolysis suggesting transmission of a cytotoxiccomponent from the BCBL-1 cell line. One Raji line cultured in 10 ng/mlTPA for 2 days, underwent an initial period of cytolysis before recoveryand resumption of logarithmic growth. This cell line (RCC-1) is amonoculture derived from Raji uncontaminated by BCBL-1 as determined byPCR amplification of HLA-DR sequences.

RCC-1 has remained positive for the KS330₂₃₃ PCR product for >6 monthsin continuous culture (approximately 70 passages), but KSHV was notdetectable by dot or Southern hybridization at any time. In situhybridization, however, with a 25 bp KSHV ORF26-derived oligomer wasused to demonstrate persistent localization of KSHV DNA to RCC-1 nuclei.As indicated in FIGS. 18A-18C, nuclei of BCBL-1 and RCC-1 (from passage˜65) cells had detectable hybridization with the ORF26 oligomer, whereasno specific hybridization occurred with parental Raji cells (FIG. 18B).KSHV sequences were detectable in 65% of BCBL-1 and 2.6% of RCC-1 cellsunder these conditions. In addition, forty-five monoclonal cultures weresubcultured by serial dilution from RCC-1 at passage 50, of which eight(18%) clones were PCR positive by KS330₂₃₃. While PCR detection usingunnested KS330₂₃₃ primer pairs was lost by passage 15 in each of theclonal cultures, persistent KSHV genome was detected in 5 clones usingtwo more sensitive nonoverlapping nested PCR primer sets [33] suggestingthat KSHV genome is lost over time in RCC-1 and its clones.

Low but persistent levels of KS330₂₃₃ PCR positivity were found for oneof four Raji, one of four Bjab, two of three Molt-3, one of one owlmonkey kidney cell lines and three of eight human fetal cord bloodlymphocyte (FCBL) cultures after inoculation with 0.2-0.45μ filteredBCBL-1 supernatants. Among the PCR positive cultures, PCR detectablegenome was lost after 2-6 weeks and multiple washings. Five FCBLcultures developed cell clusters characteristic of EBV immortalizedlymphocytes and were positive for EBV by PCR using EBER primers [23);three of these cultures were also initially KS330₂₃₃ positive. None ofthe recipient cell lines had detectable KSHV genome by dot blothybridization.

Seroloaic Studies:

Indirect immunofluorescence antibody assays (IFA) were used to assessthe presence of specific antibodies against the KSHV- and EBV-infectedcell line BHL-6 in the sera from AIDS-KS patients and control patientswith HIV infection or AIDS. BHL-6 was substituted for BCBL-1 for reasonsof convenience; preliminary studies showed no significant differences inIFA results between BHL-6 and BCBL-1. BHL-6 have diffuseimmunofluorescent cell staining with most KS patient and controlunabsorbed sera suggesting nonspecific antibody binding (FIGS. 19A-19D).After adsorption with paraformaldehyde-fixed, TPA-induced P3H3 (an EBVproducer sublime of P3J-HR1, a gift of Dr. George Miller) to removecross-reacting antibodies against EBV and lymphocyte antigens, patientsera generally showed specular nuclear staining at high titers whilethis staining pattern was absent from control patient sera (FIGS. 19Band 19D). Staining was localized primarily to the nucleus but weakcytoplasmic staining was also present at low sera dilutions.

With unadsorbed sera, the initial endpoint geometric mean titers (GMT)against BHL-6 cell antigens for the sera from AIDS-KS patients(GMT=1:1153, range: 1:150 to 1:12,150) were higher than for sera fromcontrol, non-KS patients (GMT=1:342; range 1:50 to 1:12,150; p=0.04)(FIG. 13). While AIDS-KS patients and HIV-infected gay/bisexual andintravenous drug user control patients had similar endpoint titers toBHL-6 antigens (GMT=1:1265 and GMT=1:1578, respectively), hemophilicAIDS patient titers were lower (GMT=1:104). Both case and controlpatient groups had elevated IFA titers against the EBV infected cellline P3H3.

The difference in endpoint GMT between case and control titers againstHBL-6 antigens increased after adsorption with P3H3. After adsorption,case GMT declined to 1:780 and control GMT declined to 1:81 (p=0.00009).Similar results were obtained by using BCBL-1 instead of HBL-6 cells, bypre-adsorbing with EBV-infected nonproducer Raji cells instead of P3H3and by using sera from a homosexual male KS patient without HIVinfection, in complete remission for KS for 9 months (BHL-6 titer 1:450,P3H3 titer 1:150). Paired sera taken 8-14 months prior to KS onset andafter KS onset were available for three KS patients: KS patients 8 and13 had eight-fold rises and patient 8 had a three-fold fall inP3H3-adsorbed BCBL-1 titers from pre-onset sera to post-KS sera.

Discussion

These studies demonstrate that specific DNA sequences found in KSlesions by representational difference analysis belong to a newlyidentified human herpesvirus. The current studies define this agent as ahuman gamma-2 herpesvirus that can be continuously cultured innaturally-transformed, EBV-coinfected lymphocytes from AIDS-relatedbody-cavity based lymphomas.

Sequence analysis of the KS5 lambda phage insert provides clear evidencethat the KS330Bam sequence is part of a larger herpesvirus genome. KS5has a 54.0% G+C content which is considerably higher than thecorresponding HVS region (34.3% G+C). While there is no CpG dinucleotidesuppression in the KS5 sequence, the corresponding HVS region has a 0.33expected:observed CpG dinucleotide ratio [1]. The CpG dinucleotidefrequency in herpesviruses varies from global CpG suppression amonggammaherpesviruses to local CpG suppression in the betaherpesviruses,which may result from deamination of 5′-methylcytosine residues at CpGsites resulting in TpG substitutions [21]. CpG suppression amongherpesviruses [21, 30, 44] has been hypothesized to reflectco-replication of latent genome in actively dividing host cells, but itis unknown whether or not KSHV is primarily maintained by a lyticreplication cycle in vivo.

The 20,705 bp KS5 fragment has 17 protein-coding regions, 15 of whichare complete ORFs with appropriately located TATA and polyadenylationsignals, and two incomplete ORFs located at the phage insert termini.Sixteen of these ORFs correspond by sequence and collinear positionalhomology to 15 previously identified herpesviral genes including thehighly conserved spliced gene. The conserved positional and sequencehomology for KSHV genes in this region are consistent with thepossibility that the biological behavior of the virus is similar to thatof other gammaherpesviruses. For example, identification of a thymidinekinase-like gene on KS5 implies that the agent is potentiallysusceptible to TK-activated DNA polymerase inhibitors and like otherherpesviruses possesses viral genes involved in nucleotide metabolismand DNA replication [41]. The presence of major capsid protein andglycoprotein H gene homologs suggest that replication competent viruswould produce a capsid structure similar to other herpesviruses.

Phylogenetic analyses of molecular sequences show that KSHV belongs tothe gamma-2 sublineage of the Gammaherpesvirinae subfamily, and is thusthe first human gamma-2 herpesvirus identified. Its closest knownrelative based on available sequence comparisons is HVS, a squirrelmonkey gamma-2 herpesvirus that causes fulminant polyclonal T celllymphoproliferative disorders in some New World monkey species. Data forthe gamma-2 sublineage are sparse: only three viruses (KSHV, HVS andEHV2) can at present be placed on the phylogenetic tree with precision(the sublineage also contains murine herpesvirus 68 and BHV4 [33]).Given the limitation in resolution imposed by this thin background, KSHVand HVS appear to represent a lineage of primate gamma-2 viruses.Previously, McGeoch et al. [33] proposed that lines of gamma-2herpesviruses may have originated by cospeciation with the ancestors oftheir host species. Extrapolation of this view to KSHV and HVS suggeststhat these viruses diverged at an ancient time, possiblycontemporaneously with the divergence of the Old World and New Worldprimate host lineages. Gammaherpesviruses are distinguished as asubfamily by their lymphotrophism [41] and this grouping is supported byphylogenetic analysis based on sequence data [33]. The biologic behaviorof KSHV is consistent with its phylogenetic designation in that KSHV canbe found in in vitro lymphocyte cultures and in in vivo samples oflymphocytes [3].

This band appears to be a linear form of the genome because other “highmolecular weight” bands are present for both EBV and KSHV in BCBL-1which may represent circular forms of their genomes. The linear form ofthe EBV genome, associated with replicating and packaged DNA [41]migrates substantially faster than the closed circular form associatedwith latent viral replication [24]. While the 270 kb band appears to bea linear form, it is also consistent with a replicating dimer plasmidsince the genome size of HVS is approximately 135 kb. The true size ofthe genome may only be resolved by ongoing mapping and sequencingstudies.

Replication deficient EBV mutants are common among EBV strains passagedthrough prolonged tissue culture [23]. The EBV strain infecting Raji,for example, is an BALF-2 deficient mutant [19]; virus replication isnot inducibile with TPA and its genome is maintained only as a latentcircular form [23, 33]. The EBV strain coinfecting BCBL-1 does notappear to be replication deficient because TPA induces eight-foldincreases in DNA content and has an apparent linear form on CHEFelectrophoresis. KSHV replication, however, is only marginally inducedby comparable TPA treatment indicating either insensitivity to TPAinduction or that the genome has undergone loss of genetic elementsrequired for TPA induction. Additional experiments, however, indicatethat KSHV DNA can be pelleted by high speed centrifugation of filteredorganelle-free, DNase I-protected BCBL-1 cell extracts, which isconsistent with KSHV encapsidation.

Transmission of KSHV DNA from BCBL-1 to a variety of recipient celllines is possible and KSHV DNA can be maintained at low levels inrecipient cells for up to 70 passages. However, detection of virusgenome in recipient cell lines by PCR may be due to physical associationof KSHV DNA fragments rather than true infection. This appears to beunlikely given evidence for specific nuclear localization of the ORF26sequence in RCC-1. If transmission of infectious virus from BCBL-1occurs, it is apparent that the viral genome declines in abundance withsubsequent passages of recipient cells. This is consistent with studiesof spindle cell lines derived from KS lesions. Spindle cell culturesgenerally have PCR detectable KSHV genome when first explanted, butrapidly lose viral genome after initial passages and established spindlecell cultures generally do not have detectable KSHV sequences [3].

Infections with the human herpesviruses are generally ubiquitous in thatnearly all humans are infected by early adulthood with six of the sevenpreviously identified human herpesviruses [42]. Universal infection withEBV, for example, is the primary reason for the difficulty in clearlyestablishing a causal role for this virus in EBV-associated humantumors. The serologic studies identified nuclear antigen in BCBL-1 andHBL-6 which is recognized by sera from AIDS-KS patients but generallynot by sera from control AIDS patients without KS after removal ofEBV-reactive antibodies. These data are consistent with PCR studies ofKS and control patient lymphocytes suggesting that KSHV is notubiquitous among adult humans, but is specifically associated withpersons who develop Kaposi's sarcoma. In this respect, it appears to beepidemiologically similar to HSV2 rather than the other known humanherpesviruses. An alternative possibility is that elevated IFA titersagainst BCBL-1 reflect disease status rather than infection with thevirus.

REFERENCES

-   1. Albrecht, J.-C., J. Nicholas, D. Biller, K. R. Cameron, B.    Biesinger, C. Newamn, S. Wittmann, M. A. Craxton, H. Coleman, B.    Fleckenstein, and R. W. Honess. 1992. Primary structure of the    Herpesvirus saimiri genome. J. Virol. 66:5047-5058.-   2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J.    Lipman. 1990. Basic local alignment search tool. J Mol. Biol.    215:403-410.-   3. Ambroziak, J. A., D. J. Blackbourn, B. G. Herndier, R. G.    Glogau, J. H. Gullett, A. R. McDonald, E. T. Lennette, and J. A.    Levy. 1995. Herpes-like sequences in HIV-infected and uninfected    Kaposi's sarcoma patients. Science. 268:582-583.-   4. Baer, R., A. T. Bankier, P. L. Biggin, P. L. Deininger, P. J.    Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell, C.    Séguin, P. S. Tuffnell, and B. G. Barrel. 1984. DNA sequence and    expression of the B95-8 Epstein-Barr virus genome. Nature.    310:207-211.-   5. Barton, G. J., and M. J. E. Sternberg. 1987. A strategy for the    rapid multiple alignment of protein sequences. Confidence levels    from tertiary structure comparisons. J Mol. Biol. 198:327-37.-   6. Beral, V., T. A. Peterman, R. L. Berkelman, and H. W.    Jaffe. 1990. Kaposi's sarcoma among persons with AIDS: a sexually    transmitted infection? Lancet. 335:123-128.-   7. Boshoff, C. D. Whitby, T. Hatziionnou, C. Fisher, J. van der    Walt, A. Hatzakis, R. Weiss, and T. Schulz. 1995. Kaposi's    sarcoma-associated herpesvirus in HIV-negative Kaposi's sarcoma.    Lancet. 345:1043-44.-   8. Cesarman, E., Y. Chang, P. S. Moore, J. W. Said, and D. M.    Knowles. 1995. Kaposi's sarcoma-associated herpesvirus-like DNA    sequences are present in AIDS-related body cavity based lymphomas.    New England J. Med. 332:1186-1191.-   9. Chang, Y., E. Cesarman, M. S. Pessin, F. Lee, J. Culpepper, D. M.    Knowles, and P. S. Moore. 1994. Identification of herpesvirus-like    DNA sequences in AIDS-associated Kaposi's sarcoma. Science.    265:1865-69.-   10. Chee, M. S., S. B. Bankier, C. M. Bohni, R. C. Brown, T.    Horsnell, C. A. Hutchison, T. Kouzarides, J. A. Martignetti, E.    Preddie, S. C. Satchwell, P. Tomlinson, K. M. Weston, and B. G.    Barrell. 1990. Analysis of the protein coding content of the    sequence of cytomegalovirus strain AD169. Curr Top Microbiol    Immunol. 154:125-69.-   11. Collandre, H., S. Ferris, O. Grau, L. Montagnier, and A.    Blanchard. 1995. Kaposi's sarcoma and new herpesvirus. Lancet.    345:1043.-   12. Dupin, N., M. Grandadam, V. Calvez, I. Gorin, J. T. Aubin, S.    Harvard, F. Lamy, M. Leibowitch, J. M. Huraux, J. P. Escande, and H.    Agut. 1995. Herpesvirus-like DNA in patients with Mediterranean    Kaposi's sarcoma. Lancet. 345:761-2.-   13. Feinberg, A. P., and B. Vogelstein. 1983. A technique for    radiolabeling DNA restriction endonuclease fragments to high    specific activity. Anal Biochem. 132:6.-   14. Felsenstein, J. 1989. PHYLIP-phylogeny inference package (ver    3.2). Cladistics. 5:164-6.-   15. Felsenstein, J. 1988. Phylogenies from molecular sequences:    inferences and reliability. Annual Rev Microbiol. 22:521-65.-   16. Genetics Computer Group. 1994. Program manual for the GCG    package, version 8, Madison, Wis.-   17. Gompels, U. A., J. Nicholas, G. Lawrence, M. Jones, B. J.    Thomson, M. E. D. Martin, S. Efstathiou, M. Craxton, and H. A.    Macaulay. 1995. The DNA sequence of human herpesvirus-6: Structure,    coding content and genome evolution. Virology. 209:29-51.-   18. Hatfull, G., A. T. Bankier, B. G. Barrell, and P. J.    Farrell. 1988. Sequence analysis of Raji Epstein-Barr virus DNA.    Virol. 164:334-40.-   19. Holmberg, S. D. 1990. Possible cofactors for the development of    AIDS-related neoplasms. Cancer Detection and Prevention. 14:331-336.-   20. Honess, R. W. 1984. Herpes simplex and ‘the herpes complex’:    diverse observations and a unifying hypothesis. J Gen Virol.    65:2077-2107.-   21. Honess, R. W., U. A. Gompels, B. G. Barrell, M. Craxton, K. R.    Cameron, R. Staden, Y.-N. Chang, and G. S. Hayward. 1989. Deviations    from expected frequencies of CpG dinucleotides in herpesvirus DNAs    may be diagnostic of differences in the states of their latent    genomes. J Gen Virol. 70:837-55.-   22. Hsu, D., L. M. Shih, and Y. C. Zee. 1990. Nucleotide sequence of    a 3.5 nucleotide fragment of malignant catarrhal fever virus strain    WC11. Arch Virol. 113:53-60,-   23. Kieff, E., and D. Liebowitz. 1990. Epstein-Barr virus and its    replication, p. 1889-1920. In B. N. Fields and D. M. Knipe (ed.),    Virology, vol. 2. Raven Press, New York.-   24. Kolman, J. L., C. J. Kolman, and G. Miller. 1992. Marked    variation in the size of genomic plasmids among members of the    family of related Epstein-Barr viruses. Proc Natl Acad Sci, USA.    89:7772-7776.-   25. Lebbé, C., P. de Crémoux, M. Rybojad, C. Costa da Cunha, P.    Morel, and F. Calvo. 1995. Kaposi's sarcoma and new herpesvirus.    Lancet. 345:1180.-   26. Lin, J. C., S. C. Lin, B. K. De, W. P. Chan, and B. L.    Evatt. 1993. Precision of genotyping of Epstein-Barr virus by    polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C and    EBER): predominance of type A virus associated with Hodgkin's    disease. Blood. 81:3372-81.-   27. Liu, Z., S. Yu-Kai, Y.-P. Xi, P. Harris, and N.    Suciu-Foca. 1992. T cell recognition of self-human    histocompatibility leukocyte antigens (HLA)-DR peptides in the    context of syngeneic HLA-DR molecules. J Exp Med. 175:1663-8.-   28. Lomonte, P., M. Bublot, P-P. Pastoret, and E. Thiry. 1992.    Location and characterization of the bovine herpesvirus type 4    thymidine kinase gene; comparison with thymidine kinase of other    herpesviruses. Arch. Virol. 127:327-337.-   29. Martin, M. E. D., J. Nicholas, B. J. Thomson, C. Newman,    and R. W. Honess. 1991. Identification of a transactivating function    mapping to the putative immediate-early locus of human    herpesvirus 6. J Virol. 65:5381-5390.-   30. McGeoch, D. J., and S. Cook. 1994. Molecular phylogeny of the    Alphaherpesvirinae subfamily and a proposed evolutionary timescale.    J Mol. Biol. 238:9-22.-   31. McGeoch, D. J., S. Cook, A. Dolan, F. E. Jamieson, and E. A. R.    Telford. 1995. Molecular phylogeny and evolutionary timescale for    the family of mammalian herpesviruses. J Molec Biol. 247:443-58.-   32. Miller, G. 1990. Epstein-Barr virus: Biology, pathogenesis and    medical aspects, p. 1921-1957. In B. N. Fields and D. M. Knipe    (ed.), Virology, 2nd ed, vol. 2. Raven Press, New York.-   33. Moore, P. S., and Y. Chang. 1995. Detection of herpesvirus-like    DNA sequences in Kaposi's sarcoma lesions from persons with and    without HIV infection. New England J Med. 332:1181-1185.-   34. Mukai, T., Y. Isegawa, and K. Yamanishi. 1995. Identification of    the major capsid protein gene of human herpesvirus 7. Virus Res.    37:55-62.-   35. Oettle, A. G. 1962. Geographic and racial differences in the    frequency of Kaposi's sarcoma as evidence of environmental or    genetic causes, vol. 18. Symposium on Kaposi's sarcoma: Unio    Internationalis Contra Cancrum, Karger, Basel.-   36. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for    biological sequence analysis. Proc Natl Acad Sci, USA. 85:2444-8.-   37. Peterman, T. A., H. W. Jaffe, A. E. Friedman-Kien, and R. A.    Weiss. 1991. The aetiology of Kaposi's sarcoma, p. 23-37, Cancer,    HIV, and AIDS, vol. 10. Imperial Cancer Research Fund. London.-   38. Raab-Traub, N., and K. Flynn. 1986. The structure of the termini    of the Epstein-Barr virus as a marker of clonal cellular    proliferation. Cell. 47:883-889.-   39. Roizman, B. 1993. The family Herpesviridae, p. 1-9. In B.    Roizman and R. J. Whitley and C. Lopez (ed.), The Human    Herpeviruses. Raven Press, Ltd., New York.-   40. Roizman, B. 1995. New viral footprints in Kaposi's sarcoma. N    Engl J Med. 332:1227-1228.-   41. Roizman, B., R. C. Desrosiers, B. Fleckenstein, C. Lopez, A. C.    Minson, and M. J. Studdert. 1992. The family Herpesviridae: an    update. Arch Virol. 123:425-449.-   42. Sandford, G. R., K. Ho, and W. H. Burns. 1993. Characterization    of the major locus of immediate-early genes of rat cytomegalovirus.    J Virol. 67:4093-4103.-   43. Schalling, M., M. Ekman, E. E. Kaaya, A. Linde, and P.    Bieberfeld. 1995. A role for a new herpesvirus (KSHV) in different    forms of Kaposi's sarcoma. Nature Med. 1:707-8.-   44. Schwartz, R. M., and M. O. Dayhoff. 1978. Matrices for detecting    distant relationships, p. 353-8. In M. O. Dayhoff (ed.), Atlas of    protein sequence and structure, vol. 5, supple 3. National    Biomedical Research Foundation, Washington.-   45. Su, I.-J., Y.-S. Hsu, Y.-C. Chang, and I.-W. Wang. 1995.    Herpesvirus-like DNA sequence in Kaposi's sarcoma from AIDS and    non-AIDS patients in Taiwan. Lancet. 345:722-23.-   46. Telford, E. A. R., M. S. Watson, H. C. Aird, J. Perry, and A. J.    Davison. 1995. The DNA sequence of equine herpesvirus 2. J Molec    Biol. 249:520-8.-   47. zur Hausen, H., F. J. O'Neill, and U. K. Freese. 1978.    Persisting oncogenic herpesvirus induced by the tumor promoter TPA.    Nature. 272:373-375.

EXPERIMENTAL DETAILS SECTION III

KS Patient Enrollment: Cases and controls were selected from ongoingcohort studies based on the availability of clinical information andappropriate PBMC samples. 21 homosexual or bisexual men with AIDS whodeveloped KS during their participation in prospective cohort studieswere identified [14-16]. Fourteen of these patients had paired PBMCsamples collected after KS diagnosis (median +4 months) and at leastfour months prior to KS diagnosis (median −13 months), while theremaining 7 had paired PBMC taken at the study visit immediately priorto KS diagnosis (median −3 months) and at entry into their cohort study(median −51 months prior to KS diagnosis).

Hemophilic and Homosexual/Bisexual Male AIDS Patient Control Enrollment:Two control groups of AIDS patients were examined: 23homosexual/bisexual men with AIDS followed until death who did notdevelop KS (“high risk” control group) from the Multicenter AIDS CohortStudy [16]), and 19 hemophilic men (“low risk” control group) enrolledfrom joint projects of the National Hemophilia Foundation and theCenters for Disease Control and Prevention. Of the 16 hemophiliccontrols with available follow-up information, none are known to havedeveloped KS and <2% of hemophilic AIDS patients historically develop KS[2]. For homosexual/bisexual AIDS control patients who did not developKS, paired PBMC specimens were available at entry into their cohortstudy (median −35 months prior to AIDS onset) and at the study visitimmediately prior to nonKS AIDS diagnosis (median HBL-6 months prior toAIDS onset).

DNA Extraction and Analyses: DNA from 10⁶-10⁷ PBMC in each specimen wasextracted and quantitated by spectrophotometry. Samples were prepared inphysically isolated laboratories from the laboratory where polymerasechain reaction (PCR) analyses were performed.

All samples were tested for amplifiability using primers specific foreither the HLA-DQ locus (GH26/GH27) or b-globin [18]. PCR detection ofKSHV DNA was performed as previously described [7] with the followingnested primer sets: No. 1 outer 5′-AGCACTCGCAGGGCAGTACG-3′ (SEQ IDNO:51), 5′-GACTCTTCGCTGATGAACTGG-3′ (SEQ ID NO:52); No. 1 inner5′-TCCGTGTTGTCTACGTCCAG-3′ (SEQ ID NO:53), 5′-AGCCGAAAGGATTCCACCAT-3′(SEQ ID NO:41); No. 2 outer 5′-AGGCAACGTCAGATGTGAC-3′ (SEQ ID NO:54),5′-GAAATTACCCACGAGATCGC-3′ (SEQ ID NO:42); No. 2 inner5′-CATGGGAGTACATTGTCAGGACCTC-3′ NO:55), (SEQ ID5′-GGAATTATCTCGCAGGTTGCC-3′ (SEQ ID NO:56); No. 3 outer5′-GGCGACATTCATCAACCTCAGGG-3′ (SEQ ID NO:57),5′-ATATCATCCTGTGCGTTCACGAC-3′ (SEQ ID NO:58); No. 3 inner5′-CATGGGAGTACATTGTCAGGACCTC-3′ (SEQ ID NO: 55),5′-GGAATTATCTCGCAGGTTGCC-3′ (SEQ ID NO:56). The outer primer set wasamplified for 35 cycles at 94° C. for 30 seconds, 60° C. for 1 minuteand 72° C. for 1 minute with a 5 minute final extension cycle at 72° C.One to three ml of the PCR product was added to the inner PCR reactionmixture and amplified for 25 additional cycles with a 5 minute finalextension cycle. Primary determination of sample positivity was madewith primer set No. 1 and confirmed with either primer sets 2 or 3 whichamplify nonoverlapping regions of the KSHV hypothetical major capsidgene. Sampling two portions of the KSHV genome decreased the likelihoodof intraexperimental PCR contamination. These nested primer sets are 2-3logs more sensitive for detecting KSHV sequences than the previouslypublished KS330₂₃₃ primers [6] and are estimated to be able to detect<10 copies of KSHV genome under optimal conditions. Sample preparationswere prealiquoted and amplified with alternating negative controlsamples without DNA to monitor and control possible contamination. Allsamples were tested in a blinded fashion and a determination of thepositivity/negativity made before code breaking. 35 Significance testingwas performed with Mantel-Haenszel chi-squared estimates and exactconfidence intervals using Epi-Info ver. 6 (USD Inc., Stone Mt. GA).

Results KSHV Positivity of Case and Control PBMC Samples:

Paired PBMC samples were available from each KS patient andhomosexual/bisexual control patient; a single sample was available fromeach hemophilic control patient.

To determine the KSHV positivity rate for each group of AIDS patients, asingle specimen from each participant taken closest to KS or otherAIDS-defining illness (“second sample”) was analyzed. Overall, 12 of 21(57%) of PBMC specimens from KS patients taken from 6 months prior to KSdiagnosis to 20 months after KS diagnosis were KSHV positive. There wasno apparent difference in positivity rate between immediatepre-diagnosis and post-diagnosis visit specimens (4 of 7 (57%) vs. 8 of14 (57%) respectively).

The number of KSHV positive control PBMC specimens from bothhomosexual/bisexual (second visit) and hemophilic patient controls wassignificantly lower. Only 2 of 19 (11%) hemophilic PBMC samples werepositive (odds ratio 11.3, 95% confidence interval 1.8 to 118) and only2 of 23 (9%) PBMC samples from homosexual/bisexual men who did notdevelop KS were positive (odds ratio 14.0, 95% confidence interval 2.3to 144). If all KS patient PBMC samples taken immediately prior to orafter diagnosis were truly infected, the PCR assay was at least 57%sensitive in detecting KSHV infection among PBMC samples. No significantdifferences in CD4+ counts were found for KS patients andhomosexual/bisexual patients without KS at the second sample evaluation(Kruskall-Wallis p=0.15) (FIG. 21). CD4+ counts from the single samplefrom hemophilic AIDS patients were higher than CD4+ counts from KSpatients (Kruskall-Wallis p=0.004), although both groups showed evidenceof HIV-related immunosuppression.

Longitudinal Studies:

Paired specimens were available from all 21 KS patients and 23homosexual/bisexual male AIDS control patients who did not develop KS.For the KS group, initial PBMC samples were taken four to 87 months(median 13 months) prior to the onset of KS. Initial PBMC samples fromthe control group were drawn 13 to 106 months (median 55 months) priorto onset of first nonKS AIDS-defining illness (1987 CDC surveillancedefinition). 11 of 21 (52%) of KS patients had detectable KSHV DNA inPBMC samples taken prior to KS onset compared to 2 of 19 (11%, p=0.005)hemophilic control samples, and 1 (4%, p=0.0004) and 2 (9%, p=0.002) of23 homosexual/bisexual control samples taken at the first and secondvisits respectively (FIGS. 20A-20B). The figure shows that 7 of thepaired KS patient samples were positive at both visits, 5 KS patientsand 2 control patients converted from negative to positive and two KSpatients and one control patient reverted from positive to negativebetween visits. The remaining 7 KS patients and 20 control patients werenegative at both visits.

For the 5 KS patients that converted from an initial negative PBMCresult to a positive result at or near to KS diagnosis, the medianlength of time between the first sample and the KS diagnosis was 19months. Three of the 6 KS patients that were negative at both visits hadtheir last PBMC sample drawn 2-3 months prior to onset of illness. It isunknown whether these patients became infected between their last studyvisit and the KS diagnosis date.

Discussion

Ambroziak and coworkers have found evidence that KSHV preferentiallyinfects CD19+ B cells by PBMC subset examination of three patients [19].Other gammaherpesviruses, such as Epstein-Barr virus (EBV) andherpesvirus saimiri are also lymphotrophic herpesviruses and can causelymphoproliferative disorders in primates [11, 20].

It is possible that KSHV, like most human herpesviruses, is a ubiquitousinfection of adults [21]. EBV, for example, is detectable by PCR inCD19+ B lymphocytes from virtually all seropositive persons [22] andapproximately 98% MACS study participants had EBV VCA antibodies atentry into the cohort study [23]. The findings, however, are mostconsistent with control patients having lower KSHV infection rates thancases and that KSHV is specifically associated with the subsequentdevelopment of KS. While it is possible that control patients areinfected but have an undetectably low KSHV viral PBMC load, theinability to find evidence of infection in control patients under avariety of PCR conditions suggests that the majority of control patientsare not infected. Nonetheless, approximately 10% of these patients wereKSHV infected and did not develop KS. It is unknown whether or not thisis similar to the KSHV infection rate for the general human population.

This study demonstrates that KSHV infection is both strongly associatedwith KS and precedes onset of disease in the majority of patients. 57%of KS patients had detectable KSHV infection at their second follow-upvisit (52% prior to the onset of KS] compared to only 9% ofhomosexual/bisexual (p=0.002) and 11% of hemophilic control patients(p=0.005). Despite similar CD4+ levels between homosexual/bisexual KScases and controls, KSHV DNA positivity rates were significantly higherfor cases at both the first (p=0.005) and second sample visitsindicating that immunosuppression alone was not responsible for theseelevated detection rates. It is also unlikely that KSHV simply colonizesexisting KS lesions in AIDS patients since neither patient group had KSat the time the initial sample was obtained. Five KS patients and twohomosexual/bisexual control patients converted from a negative to apositive, possibly due to new infection acquired during the studyperiod.

The findings are in contrast to PCR detection of KSHV DNA in all 10 PBMCsamples from KS patients by Ambroziak at al. [19]. It is possible thatthe assay was not sensitive enough to detect virus in all samples sinceit was required that each positive sample to be repeatedly positive bytwo independent primers in blinded PCR assays. This appears unlikely,however, given the sensitivity of the PCR nested primer sets. The 7 KSpatients who were persistently negative on both paired samples mayrepresent an aviremic or low viral load subpopulation of KS patients.The PCR conditions test a DNA amount equivalent to approximately 2×10³lymphocytes; an average viral load less than 1 copy per 2×10³ cells maybe negative in the assay. Two KS patients and a homosexual/bisexualcontrol patient initially positive for KSHV PCR amplification revertedto negative in samples drawn after diagnosis. These results probablyreflect inability to detect KSHV DNA in peripheral blood rather thantrue loss of infection although more detailed studies of the naturalhistory of infection are needed.

The study was designed to answer the fundamental question of whether ornot infection with KSHV precedes development of the KS phenotype. Thefindings indicate that there is a strong antecedent association betweenKSHV infection and KS. This temporal relationship is an absoluterequirement for establishing that KSHV is central to the causal pathwayfor developing KS. This study contributes additional evidence for apossible causal role for this virus in the development of KS.

REFERENCES

-   1. Katz M H, Hessol N A, Buchbinder S P, Hirozawa A, O'Malley P,    Holmberg S D. Temporal trends of opportunistic infections and    malignancies in homosexual men with AIDS. J Infect Dis. 1994;    170:198-202.-   2. Beral V, Peterman T A, Berkelman R L, Jaffe H W. Kaposi's sarcoma    among persons with AIDS: a sexually transmitted infection?Lancet.    1990; 335:123-128.-   3. Archibald C P, Schechter M T, Le T N, Craib K J P, Montaner J S    G, O'Shaughnessy M V. Evidence for a sexually transmitted cofactor    for AIDS-related Kaposi's sarcoma in a cohort of homosexual men.    Epidemiol. 1992; 3:203-209.-   4. Beral V, Bull D, Jaffe H, Evans B, Gill N, Tillett H et al. Is    risk of Kaposi's sarcoma in AIDS patients in Britain increased if    sexual partners came from United States or Africa?BMJ. 1991;    302:624-5.-   5. Beral V. Epidemiology of Kaposi's sarcoma. Cancer, HIV and AIDS.    London: Imperial Cancer Research Fund; 1991:5-22.-   6. Chang Y, Cesarman E, Pessin M S, Lee F, Culpepper J, Knowles D M,    et al. Identification of herpesvirus-like DNA sequences in    AIDS-associated Kaposi's sarcoma. Science. 1994; 265:1865-69.-   7. Moore P S, Chang Y. Detection of herpesvirus-like DNA sequences    in Kaposi's sarcoma lesions from persons with and without HIV    infection. New England J Med. 1995; 332:1181-1185.-   8. Boshoff C, Whitby D, Hatziionnou T, Fisher C, van der Walt J,    Hatzakis A et al. Kaposi's sarcoma-associated herpesvirus in    HIV-negative Kaposi's sarcoma. Lancet. 1995; 345:1043-44.-   9. Su I-J, Hsu Y-S, Chang Y-C, Wang I-W. Herpesvirus-like DNA    sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in    Taiwan. Lancet. 1995; 345:722-23.-   10. Dupin N, Grandadam M, Calvez V, Gorin I, Aubin J T, Harvard S,    et al. Herpesvirus-like DNA in patients with Mediterranean Kaposi's    sarcoma. Lancet. 1995; 345:761-2.-   11. Miller G. Oncogenicity of Epstein-Barr virus. J Infect Dis.    1974; 130:187-205.-   12. Hill A B. Environment and disease: association or causation?    Proc Roy Soc Med. 1965; 58:295-300.-   13. Susser M. Judgment and causal inference: criteria in    epidemiologic studies. An J. Epid. 1977; 105:1-15.-   14. Fishbein D B, Kaplan J E, Spira T J, Miller B, Schonberger L B,    Pinsky P F, et al. Unexplained lymphadenopathy in homosexual men: a    longitudinal study. JAMA. 1985; 254:930-5.-   15. Holmberg S D. Possible cofactors for the development of    AIDS-related neoplasms. Cancer Detection and Prevention. 1990;    14:331-336.-   16. Kaslow R A, Ostrow D G, Detels R, Phair J P, Polk B F, Rinaldo    C R. The Multicenter AIDS Cohort Study: rationale, organization and    selected characteristics of the participants. Am J Epidemiol. 1987;    126:310-318.-   17. Wolinsky S. Rinaldo C, Kwok S, Sinsky J, Gupta P. Imagawa D, et    al. Human immunodeficiency virus type 1 (HIV-1) infection a median    of 18 months before a diagnostic Western blot. Ann Internal Med.    1989; 111:961.-   18. Bauer H M, Ting Y, Greer C E, Chambers J C, Tashiro C J, Chimera    J, et al. Genital papillomavirus infection in female university    students as determined by a PCR-based method. JAMA. 1991;    265:2809-10.-   19. Ambroziak J A, Blackbourn D J, Herndier B G, Glogau R G, Gullett    J H, McDonald A R, at al. Herpes-like sequences in HIV-infected and    uninfected Kaposi's sarcoma patients. Science. 1995; 268:582-583.-   20. Roizman B. The family Herpesviridae. In: Roizman B, Whitley R J,    Lopez C, eds. The Human Herpeviruses. New York: Raven Press, Ltd.;    1993:1-9.-   21, Roizman B. New viral footprints in Kaposi's sarcoma. N Engl J    Med. 1995; 332:1227-1228.-   22. Miyashita E M, Yang B, Lam K M C, Crawford D H, Thorley-Lawson    D A. A novel form of Epstein-Barr virus latency in normal B cells in    vivo. Cell. 1995; 80:593-601.-   23. Rinaldo C R, Kingsley L A, Lyter D W, Rabin B S, Atchison R W,    Bodner A J, at al. Association of HTLV-III with Epstein-Barr virus    infection and abnormalities of T lymphocytes in homosexual men. J    Infect Dis. 1986; 154:556-61.

EXPERIMENTAL DETAILS SECTION IV

To determine if the KHV-KS virus is also present in both endemic andHIV-associated KS lesions from African patients, formalin-fixed,paraffin-embedded tissues from both HIV seropositive and HIVseropositive Ugandan KS patients were compared to cancer tissues frompatients without KS in a blinded case-control study.

Patient Enrollment: Archival KS biopsy specimens were selected fromapproximately equal numbers of HIV-associated and endemic HIV-negativeKS patients enrolled in an ongoing case-control study of cancer and HIVinfection at Makerere University, Kampala Uganda. Control tissues wereconsecutive archival biopsies from patients with various malignanciesenrolled in the same study, chosen without prior knowledge of HIVserostatus. All patients were tested for HIV antibody (measured byCambridge Bioscience Recombigen Elisa assay).

Tissue preparation: Each sample examined was from an individual patient.Approximately ten tissue sections were cut (10 micron) from eachparaffin block using a cleaned knife blade for each specimen. Tissuesections were deparaffinized by extracting the sections twice with 1 mlxylene for 15 min. followed by two extractions with 100% ethanol for 15min. The remaining pellet was then resuspended and incubated overnightat 50° C. in 0.5 ml of lysis buffer (25 mM KCl, 10 mM Tris-HCl, pH 8.3,1.4 mM MgCl₂, 0.01% gelatin, 1 mg/ml proteinase K). DNA was extractedwith phenol/chloroform, ethanol precipitated and resuspended in 10 mMTris-HCl, 0.1 mM EDTA, pH 8.3.

PCR Amplification: 0.2-0.4 ug of DNA was used in PCR reactions withKS330₂₃₃ primers as previously described [7]. The samples which werenegative were retested by nested PCR amplification, which isapproximately 10²-10³ fold more sensitive in detecting KS330₂₃₃ sequencethan the previously published KS330₂₃₃ primer set [7]. These sampleswere tested twice and samples showing discordant results were retested athird time. 51 of 74 samples initially examined were available forindependent extraction and testing at Chester Beatty Laboratories,London using identical nested PCR primers and conditions to ensurefidelity of the PCR results. Results from eight samples were discordantbetween laboratories and were removed from the analysis asuninterpretable (four positive samples from each laboratory).Statistical comparisons were made using EPI-INFO ver. 5 (USD, Stone Mt.GA, USA) with exact confidence intervals.

Results:

Of 66 tissues examined, 24 were from AIDS-KS cases, 20 were from endemicHIV seronegative KS cases, and 22 were from cancer control patientswithout KS. Seven of the cancer control patients were HIV seropositiveand 15 were HIV seronegative (FIG. 22). Tumors examined in the controlgroup included carcinomas of the breast, ovaries, rectum, stomach, andcolon, fibrosarcoma, lymphocytic lymphomas, Hodgkin's lymphomas,choriocarcinoma and anaplastic carcinoma of unknown primary site. Themedian age of AIDS-KS patients was 29 years (range 3-50) compared to 36years (range 3-79) for endemic KS patients and 38 years (range 21-73)for cancer controls.

Among KS lesions, 39 of 44 (89%) were positive for KS330₂₃₃ PCR product,including KS tissues from 22 of 24 (92%) HIV seropositive and 17 of 20(85%) HIV seronegative patients. In comparison, 3 of 22 (14%) nonKScancer control tissues were positive, including 1 of 7 (14%) HIVseropositive and 2 of 15 (13%) HIV seronegative control patients (FIG.19). These control patients included a 73 year old HIV seronegative maleand a 29 year old HIV seronegative female with breast carcinomas, and a36 year old HIV seropositive female with ovarian carcinoma. The oddsratios for detecting the sequences in tissues from HIV seropositive andHIV seronegative cases and controls was 66 (95% confidence interval (95%C.I.) 3.8-3161) and 36.8 (95% C.I. 4.3-428) respectively. The overallweighted Mantel-Haenzel odds ratio stratified by HIV serostatus was 49.2(95% C.I. 9.1-335). KS tissues from four HIV seropositive children (ages3, 5, 6, and 7 years) and four HIV seronegative children (ages 3, 4, 4,and 12 years) were all positive for KS330₂₃₃.

All discordant results (i.e. KSHV negative KS or KSHV positive nonKScancers) were reviewed microscopically. All KS330₂₃₃ PCR negative KSsamples were confirmed to be KS. Likewise, all KS330₂₃₃ PCR positivenonKS cancers were found not to have occult KS histopathologically.

Discussion

These results indicate that KSHV DNA sequences are found not only inAIDS-KS [5], classical KS [6] and transplant KS [7] but also in AfricanKS from both HIV seropositive and seronegative patients. Despitedifferences in clinical and epidemiological features, KSHV DNA sequencesare present in all major clinical subtypes of KS from widely dispersedgeographic settings.

This study was performed on banked, formalin-fixed tissues whichprevented the use of specific detection assays such as Southernhybridization. DNA extracted after such treatment is often fragmentedwhich reduces the detection sensitivity of PCR and may account for the 5PCR negative KS samples found in the study. The results, however, areunlikely to be due to PCR contamination or nonspecific amplification.Specimens were tested blindly and a subset of samples were independentlyextracted and tested at a physically separate laboratory. Specimenblinding is essential to ensure the integrity of results based solely onPCR analyses. A subset of amplicons was sequenced and found to be morethan 98% identical to the published KS330₂₃₃ sequence confirming theirspecific nature and, because of minor sequence variation, making thepossibility of contamination unlikely.

In contrast to previous studies in North American and Europeanpopulations, it was found 3 of 22 control tissues to have evidence ofKSHV infection. Since these cancers represent a variety of tissue types,it is unlikely that KSHV has an etiologic role in these tumors. Onepossible explanation for the findings is that these results reflect therate of KSHV infection in the nonKS population in Uganda. Fourindependent controlled studies from North America [5 and 9] Europe [7]and Asia [8] have failed to detect evidence of KSHV infection in over200 cancer control tissues, with the exception of an unusualAIDS-associated, body-cavity-based lymphoma [9]. Taken together, thesestudies indicate that DNA-based detection of KSHV infection is rare inmost nonKS cancer tissues from developed countries. KSHV infection hasbeen reported in post-transplant skin tumors, although well-controlledstudies are needed to confirm that these findings are not due to PCRcontamination [10]. Since the rate of HIV-negative KS is much morefrequent in Uganda than the United States, detection of KSHV in controltissues from cancer patients in the study may reflect a relatively highprevalence infection in the general Ugandan population.

While KS is extremely rare among children in developed countries [2],the rate of KS in Ugandan children has risen dramatically over the past3 decades: age-standardized rates (per 100,000) for boys age 0-14 yearswere 0.25 in 1964-68 and 10.1 in 1992-93. Detection of KSHV genome in KSlesions from prepubertal children suggests that the virus has anonsexual mode of transmission among Ugandan children. That five ofthese children were 5 years old or less raises the possibility that theagent can be transmitted perinatally. Whether or not immune tolerancedue to perinatal transmission accounts for the more fulminant form of KSoccurring in African children remains to be investigated.

REFERENCES

-   1. Oettle A. G. Geographic and racial differences in the frequency    of Kaposi's sarcoma as evidence of environmental or genetic causes.    Acta Un Int Cancer 1962; 18:330-363.-   2. Beral V. Epidemiology of Kaposi's sarcoma. In: Cancer, HIV and    AIDS. London: Imperial Cancer Research Fund, 1991: 5-22.-   3. Wabinga H. R., Parkin D. M., Wabwire-Mangen F., Mugerwa J. Cancer    in Kampala, Uganda, in 1989-91: changes in incidence in the era of    AIDS. Int J Cancer 1993; 54:26-36.-   4. Kestens L. et al. Endemic Kaposi's sarcoma is not associated with    immunodeficiency. Int. J. Cancer 1985; 36:49-54.-   5. Chang Y. et al. Identification of herpesvirus-like DNA sequences    in AIDS-associated Kaposi's sarcoma. Science 1994; 266:1865-9.-   6. Moore P. S. and Chang Y. Detection of herpesvirus-like DNA    sequences in Kaposi's sarcoma lesions from persons with and without    HIV infection. New England J Med 1995; 332:1181-85.-   7. Boshoff C. et al. Kaposi's sarcoma-associated herpesvirus in HIV    negative Kaposi's sarcoma (letter). Lancet 1995; 345:1043-44.-   8. Su, I.-J., Hsu, Y.-S., Chang, Y.-C., Wang, I.-W. Herpesvirus-like    DNA sequence in Kaposi's sarcoma from AIDS and non-AIDS patients in    Taiwan. Lancet 1995; 345: 722-3.-   9. Cesarman E., Chang Y. Moore P. S. Said J. W., Knowles D. M.    Kaposi's sarcoma-associated herpesvirus-like DNA sequences are    present in AIDS-related body cavity based lymphomas. New England J    Med 1995; 332:1186-1191.-   10. Rady P. L., et al. Herpesvirus-like DNA sequences in nonKaposi's    sarcoma skin lesions of transplant patients. Lancet 1995;    345:1339-40.

EXPERIMENTAL DETAILS SECTION V Serologic Marker for KSHV InfectionMethods

Patients Serum was collected from a convenience sample of 89HIV-infected patients seen at several clinical sites in Connecticut, NewYork, and California. Demographic and clinical information was recordedon standardized forms which were linked to samples by a numerical code.Patients were classified as having KS if the diagnosis washistologically confirmed or, in the opinion of the primary clinician,the diagnosis of KS was unequivocal on clinical grounds. Eighty six(97%) were male; 90 of the 86 men (93%) were homosexual or bisexual.Forty seven patients, all male, had KS. The characteristics of the studypopulation are found in FIG. 23].

Cell lines The BCBL-1 line was established from an AIDS-associated bodycavity B cell non-Hodgkin's lymphoma [30]. Neither BCBL-1 cells, nor thetumor from which they were derived, express surface immunoglobulin or Bcell specific surface markers; however BCBL-1 cells containimmunoglobulin gene rearrangements that are characteristic of B cells[31]. KSHV DNA sequences can be detected in BCBL-1 cells by DNArepresentational difference analysis [23,32]. BCBL-1 cells also containan EBV genome detectable with several different EBV DNA probes. B95-8 isan EBV producer marmoset cell line that can be efficiently induced intoEBV lytic cycle gene expression by phorbol esters (TPA) [33,34], HH514-16 is an EBV containing cell line, originally from a Burkittlymphoma, that is optimally inducible into EBV lytic cycle geneexpression by n-butyrate [35,36]. B141 is an EBV-negative Burkittlymphoma cell line [37]. B95-8, HH514-16 and BL41 do not hybridize withthe KSHV probes. All cell lines were cultured in RPMI 1640 mediumcontaining 8% fetal calf serum.

Immunoblotting Assays Extracts of uninduced BCBL-1 cells or BCBL-1 cellsthat had been treated with 20 ng/ml TPA and 3 mM n-butyrate for 48 hrswere prepared by sonication. HH514-16 cells, treated similarly, servedto control for antibody reactivity to EBV polypeptides. Each lane of a10% or 12% polyacrylamide gel was loaded with extract of 5×10⁵ cells inSDS sample buffer; electrophoresis, transfer to nitrocellulose andblocking with skim milk followed standard protocols [38]. Sera werescreened at 1:100 dilution. The reaction was developed by 1.0 μCi of¹²⁵I Staphylococcal protein A. Radioautographs were exposed to film for24-48 hrs. Immunoblotting assays were performed and interpreted on codedsera.

Immunofluorescent assay The antigens were BCBL-1 cells that wereuntreated or treated with 3 mM n-butyrate for 48 hrs. Cells were droppedonto slides that were fixed in acetone and methanol. Sera were tested at1:10 dilution, followed by 1:30 dilution of fluoresceinated goatanti-human Ig. The reactivity of a serum was compared on untreated andn-butyrate treated BCBL-1 cells. Reactivity with 30-50% of thechemically treated BCBL-1 cells was considered a positive reaction. Allimmunofluorescence tests were performed on coded sera. The two readerswere blinded to disease status or results of immunoblotting assays.

Results

Chemical Induction of lytic cycle KSHV proteins in BCBL cells: Initialexperiments using the immunoblotting technique were designed todetermine whether BCBL-1 cells expressed unique antigenic polypeptidesthat might be specific for KSHV infection. Since sera from HIV-1infected patients with or without KS would be expected to containantibodies to EBV polypeptides and since BCBL-1 cells are duallyinfected with KSHV and EBV it was essential to distinguish EBVpolypeptides from those encoded or induced by KSHV. FIGS. 27A-27B, animmunoblot prepared from BCBL-1 cells reacted with a reference EBVantiserum, shows that BCBL-1 cells expressed two polypeptides,representing the latent nuclear antigen EBNA1 and p21, a late antigencomplex [39], that were present in other EBV producer cell lines, suchas B95-8 (FIG. 27A) and HH514-16 (FIG. 278 and FIGS. 28A-28D). When serafrom patients with KS were used as a source of antibody they failed toidentify in extracts from untreated BCBL-1 cells additional antigenicpolypeptides that were not also seen in the EBV producer cell lines.However, if extracts were prepared from BCBL-1 cells that had first beentreated with a combination of phorbol ester, TPA, and n-butyrate, KSpatient sera now recognized a number of novel polypeptides that werepresent in the BCBL-1 cell line but not in standard EBV producer celllines (FIG. 27B). The molecular weights of the most prominent of thesemany polypeptides were estimated at about 27 KDa, 40 KDa and 60 KDa on10% polyacrylamide gels. These polypeptides were detected within 24 hrsafter addition of the chemical inducing agents, but were not evident inBCBL-1 held in culture for as long as 5 days without chemical treatment.Further experiments showed that n-butyrate was the chemical agentprimarily responsible for induction of p40, whereas p60 could be inducedby TPA or n-butyrate (FIGS. 28A-28D). Since p27, p40 and p60 were notdetected in untreated cells and appeared after treatment with chemicalsthey likely represented lytic cycle rather than latent cyclepolypeptides of KSHV.

p40 and p60 are KSHV specific: FIGS. 27A-27B shows that antigenicpolypeptides corresponding in molecular weight to p40 were not observedin two EBV producer lines, B95-8 and HH514-16, that were induced intothe EBV lytic cycle by the same chemicals or in comparably treatedEBV-negative BL41 cells. Furthermore n-butyrate strongly inducedexpression of p40 in BCBL-1 cells but had little or no effect on thelevel of expression of the EBV p21 complex in the same cells. In relatedexperiments it was found that n-butyrate also induced an increase in theabundance of KSHV DNA and KSHV lytic cycle mRNA. TPA, by contrast,induced the EBV lytic cycle efficiently’ treatment with TPA caused anincrease in the abundance of the EBV p21 protein and minimal inductionof KSHV p40. These findings suggested that latency to lytic cycle switchof the two gamma herpes viruses carried by BCBL-1 cells was underseparate control and that the p40 complex was specific to the KSHVgenome.

p40 as a serologic marker for KSHV: While a few highly reactive sera,such as KS 01-03, (FIG. 27B) recognized multiple antigenic proteinsunique to the chemically induced BCBL-1 cells, including p27, p60 andp40, sera from other patients with KS did not react with p27 or p60 butstill recognized p40 (FIGS. 28A and 28B). Therefore recognition of p40was investigated as a serologic marker for infection with KSHV. Serafrom 89 HIV-1 infected patients from Connecticut, New York andCalifornia were examined for presence of antibodies to p40; only 3 of 42patients (7%) without KS had antibodies to p40 (p<0.0001 by Chi square).These three patients were homosexual or bisexual men from New York city.The positive and negative predictive values of the serologic marker forthe presence of KS were 84% and 78% respectively. Three HIV-1 infectedmen from New York with non-Hodgkin's lymphoma but without KS werenon-reactive to the KSHV p40 antigen. FIG. 25 compares the patients withKS whose serum did or did not contain antibodies to KSHV p40. NeitherCD4 cell number nor the extent of KS disease predicted the presence orabsence of a serologic response to p40.

Immunofluorescence assays: Immunoblots showed that n-butyrate inducedexpression of KSHV lytic cycle polypeptides in BCBL-1 cells withoutsignificantly affecting expression of EBV polypeptides (FIG. 28A).Therefore it was reasoned that n-butyrate might also induce many moreBCBL-1 cells into the KSHV lytic cycle than into the EBV lytic cycle.Using indirect immunofluorescence with a reference human antiserum, R Mlin FIG. 27B, that contains antibodies to EBV but not KSHV there wereabout 2% antigen positive untreated BCBL-1 cells and a similar number ofantigen positive BCBL cell that had been treated with n-butyrate. Serum01-03 that is EBV-positive and KS-positive (FIG. 27B) detected 2%antigen positive cells in the untreated BCBL population, presumably theEBV expressing cells, while it detected 50% antigen positive BCBL-1cells that had been treated with n-butyrate. This increase in the numberof antigen positive BCBL-1 cells among the n-butyrate treated populationserved as the basis of an immunofluorescence screening assay forantibodies to KSHV lytic cycle antigens (FIGS. 29A-29F). The results ofthe immunofluorescence assay were nearly identical to the immunoblottingassay (FIG. 26). Among 89 sera there were only 4 (3%) that werediscordant in the two assays. Three sera scored positive by IFA andnegative by immunoblotting: one was considered positive byimmunoblotting and negative by IFA. 68% of patients with KS and 12% ofHIV-1 infected patients without KS were reactive by indirectimmunofluorescence assay (IFA). Thus using two different assays,antibodies to KSHV lytic cycle antigens were found 6 to 9 times morefrequently among patients with KS than among HIV-1 infected patientswithout KS. Stated another way, among individuals who were seropositiveto KSHV p40 32/35 (91%) had KS. Among those seropositive by theimmunofluorescence assay 32/37 (86%) had KS. Thus infection with KSHV,as defined by these serologic markers, carries a high risk ofdevelopment of KS.

Discussion

The recent discovery of genetic sequences representative of a new humanherpes virus in KS tumor tissue, taken together with past epidemiologicobservations, strongly implicate this novel agent in the pathogenesis ofKS. However, these observations, by themselves, do not permit theconstruction of a unified theory of pathogenesis that accounts for themany mysterious features of KS. For example, the relative contributionof HIV-1, other forms of immunosuppression, geographic factors, sexdifferences, the role of cytokines and growth factors, and theoccurrence of distinct clinical variants must all be eventuallyunderstood. By identifying the infection rate in different populations aserologic marker for infection with KSHV would be great aid inunraveling the significance of the new virus in this complicated puzzle.

One possibility is that KSHV, the putative etiologic agent is, like allthe other human herpes viruses, a ubiquitous, or at least widespreadvirus which infects large segments of the human population. Individualswho are immunosuppressed would have a greater likelihood of developingdisease, whereas immunocompetent individuals would remain healthy. Thispathogenetic model is similar to that postulated for the role that EBVplays in non-Hodgkin's lymphoma or cytomegalovirus in retinitis inpatients with AIDS. If this model is correct a very high proportion ofthe adult human population might be found to be seropositive for KSHV.The model of a ubiquitous virus selectively causing disease inimmunodeficient individuals does not account for classical KS affectingpatients who are not immunocompromised nor does it account for theobservations that endemic KS in Africa preceded the HIV-1 epidemic.Since many African patients with KS are HIV-1 negative other co-factorsmust be implicated.

The other possibility is that KSHV infection occurs selectively in thehuman population. Transmission may be promoted by sexual behavior thatalso carries a high risk of acquiring HIV-1. In this scenarioseroprevalence of KSHV would be expected to be higher in HIV-1seropositive and HIV-1 seronegative homosexual men than in otherpopulations. If the virus alone were capable of inducing disease,acquisition of KSHV infection, as monitored by the presence of antibody,would be associated with a high rate of clinically evident KS. However,if KSHV infection needed to accompanied by other co-factors to causedisease, the prevalence of antibody of KSHV might be similar amongpatients with and without KS. The other co-factors would not beidentified in a serologic test for antibodies to KSHV antigens.

The findings, using tests for antibodies to KSHV lytic cycle antigens,are consistent with the general model in which infection with KSHV isinfrequent but associated with a high rate of apparent disease. Only afew HIV-1 infected patients without KS had antibodies to the KSHV lyticcycle antigens; by contrast a very high proportion of HIV-1 infected menwho had clinically evident KS were seropositive. This finding suggeststhat a high proportion of individuals who are dually infected with HIV-1and KSHV develop KS. However, another interpretation of the data ispossible, though this interpretation is novel and no other examples areknown among the human herpes virus family. Infection with KSHV might beubiquitous, antibodies to the virus would not normally be detected inhealthy infected individuals. Antibodies would only appear after thevirus has been reactivated from the latent into the lytic cycle as mightoccur during the course of immunosuppression. Thus the two serologictests that are described would indicate reactivated infection but wouldnot be an index of past exposure to the virus. If this interpretation iscorrect, it should be possible to demonstrate KSHV DNA sequences or totisolate the virus from healthy individuals who are KSHV seronegative.

Regardless of which of these two interpretations is correct, theserologic studies provide a strong correlation between the presence ofantibodies to KSHV lytic cycle gene products and clinical KS.Nonetheless there are two groups of patients whose serologic resultsrequire further explanation. One group consists of the few patients withpositive serology for KSHV p40 without clinical KS. They may havesubclinical or visceral disease, or they may develop KS in the future.The other group is the approximately 30% of patients with KS whose seralacked antibody to p40. The patients with KS who were p40 seronegativewere not misclassified since the diagnosis was confirmed in all of themby biopsy (FIG. 25). It is possible that the antibodies being measuredare variable and wax and wane with time following infection. Theappearance of antibody to p40 may reflect the extent of lytic viralreplication which may vary during different phases of the disease. Todetermine whether this is true prospective studies including serialbleedings are required.

p40 is likely to be only one among a number of KSHV antigens recognizedby the infected patients. Antibody recognition of other KSHV antigensmay not be possible on immunoblots because they comigrate with EBVpolypeptides, because the BCBL-1 cells cannot be induced to expressthese antigens, or because the antigens are of low abundance ordenatured on the immunoblots. In some individuals serum antibodies top40 may be consumed in immune complexes with p40 antigen in thecirculation. Thus detection of p40 on immunoblots may not be of optimalsensitivity. In this connection three sera recognized antigens inimmunofluorescence tests but did not react with p40 on western blots.The serologic test employing whole BCBL-1 cells as antigen are clearlyfirst generation assays to be improved by better characterization of theKSHV gene products and preparation of recombinant antigens.

Lack of a serologic response to p40 could also reflect severely impairedhumoral immunity. Although humoral immunity is usually relatively intactin HIV infection, examples of impaired antibody response have beendescribed. For instance, some individuals are known to have impairedantibody responses to parvovirus B19(40 and others have been observed tolose antibodies to hepatitis B surface antigen (41]. An associationbetween the degree of immunosuppression, as monitored by the number ofCD4 cells, and the presence or absence of antibody p40 among patientswith KS was not found (FIG. 25). Furthermore all the patients with orwithout antibodies to KSHV p40 had antibodies to EBV p21 suggesting anintact humoral immune response.

In these serologic studies, as in the genetic probe studies previouslyreported, KSHV infection was found in the majority, but not all,patients with KS. Assuming that methodologic explanations do not accountexclusively for the seronegative patients, other pathways, in additionto infection with KSHV, may lead to development of KS. In fact, mostdata suggest that the pathogenesis of KS is a multifactorial process. Ithas been observed that the product of the HIV-tat gene stimulates growthof KS tissue culture cells [42] and can induce KS-like lesions in mice[43]. These findings suggest a direct role for HIV-1 in the pathogenesisof KS, at least in HIV-infected hosts. In other settings, other growthfactors may play a similar or complementary function. Interleukin-6 andbasic fibroblast growth factor are both known stimulate growth of KScells in vitro [44]. Interleukin-6 is also produced in AIDS-KS derivedcell culture [44]. Thus, KS pathogenesis may involve autocrine andparacrine growth factors together with infection with KSHV in somepatients or with certain strains of HIV-1 in other patients. Ifinfection with KSHV is the sine qua non of this process on would expectto see evidence of KSHV infection in all patients with KS.

In summary, an immunoblotting and a immunofluorescence screening assayfor detection of antibodies to lytic cycle antigens of KSHV isdisclosed. These assays should permit detailed seroepidemiologicinvestigations of KSHV. The findings support the notion of a strongassociation between infection with KSHV and the development of KS inHIV-infected patients. Infection with KSHV, as defined by theseserologic assays, appears to carry an extremely high risk of developmentof clinical KS.

REFERENCES

-   1. Kaposi M. Idiopathic multiple pigmented sarcoma of the skin.    Cancer 1892; 31:3-   2. Safai B, Good R A. Kaposi's sarcoma: A review and recent    developments. Clin Bull 1980; 10:62-8.-   3. Loethe R. Kaposi's sarcoma in Ugandan Africans. Acta Pathol    Microbiol Scand 1963 (suppl); 161:1-70.-   4. Gordon J A. Clinical features of Kaposi's sarcoma amongst    Rhodesian Africans. Central African Journal of Medicine 1972;    19:1-6.-   5. Penn I. Kaposi's sarcoma in organ transplant recipients.    Transplantation 1979; 27:8-1.-   6. Friedman-Kien A E. Kaposi's sarcoma and Pneumocystis carinii    pneumonia among homosexual men—New York City and California. MMWR.    1981; 30:305-8.-   7. Friedman-Kien A E, Laubensin L J, Rubenstein P, et al.    Disseminated Kaposi's sarcoma in homosexual men. Ann Intern Med    1982; 96:693-700.-   8. Berel V, Peterman T A, Berkel R L, Jaffe H W. Kaposi's sarcoma    among persons with AIDS: a sexually transmitted disease? Lancet    1990; 335:123-128.-   9. Drew W L, Mills J, Hauer L B, et al. Declining prevalence of    Kaposi's sarcoma in homosexual AIDS patients paralleled by a fall in    cytomegalovirus transmission. Lancet 1988; 66.-   10. Beral V, Peterman T A, Berkelman R L, et al. Kaposi's sarcoma    among persons with AIDS: A sexually transmitted infection? Lancet    1990; 2:123.-   11. Barley A C, Downng R G, Chemgson-Popov R et al. HTLV-III    serology distinguishes atypical and endemic Kaposi's sarcoma in    Africa. Lancet 1985; 1:359.-   12. Ziegler, J. L. J. A. Beckstead, P. A. Volberding, et al. 1984.    Non-Hodgkin's lymphoma in 90 homosexual men: Relation to generalized    lymphoadenopathy and the acquired immunodeficiency syndrome. N.    Engl. J. Med. 311:565-570.-   13. Levine A M. Non-Hodgkins lymphomas and other malignancies in the    acquired immunodeficiency syndrome. Semin Oncol 1987; 14:34-9.-   14. Knowles D M, Chamulak M, Subar M, et al. Lymphoid neoplasia    associated with the acquired immunodeficiency syndrome. Ann Intern    Med 1988; 108:744-53.-   15. MacMahon E M E, Glass J D, Hayward S D, Mann R B, Becher P S,    Charache P, McArthur J C, Ambender R F. Epstein-Barr virus in    AIDS-related primary central nervous system lymphoma. Lancet 1991;    338:969-53.-   16. Sillman F H, Sedlis A. Anogenital papillomavirus infection and    neoplasia in immunodeficiency women. Obstet Gynecol Clin North Am    1987; 260:348-53.-   17. Drew W L, Mills J. Hauer L B, Miner R C, et al. Cytomegalovirus    and Kaposi's sarcoma in young homosexual men. Lancet. 1982;    2:125-127.-   18. Callant J E, Moore R D, Richman D D, Keruly J, Chaisson R E.    Risk factors for Kaposi's sarcoma in patients with advanced human    immunodeficiency virus disease treated with zidovudine. Arch Inter    Med 1994; 154:566-72.-   19. Giraldo, G., E. Beth, and E. Huang. 1980. Kaposi's sarcoma and    its relationship to cytomegalovirus III CMV DNA and CMV early    antigens in Kaposi's sarcoma. Int. J. Cancer 26:23-29.-   20. Ambinder, R. F., Newman, C. Hawyard, G. S. et al. 1987 Lack of    association of cytomegalovirus with endemic African Kaposi's    sarcoma J. Inf. Dis. 156:193-7.-   21. Jahan N, Razzaque A, Greenspan J et al. Analysis of human KS    biopsies and cloned cell lines for cytomegalovirus, HIV-1 and other    selected DNA virus sequences. ADIS Res Human Retrovirus 1989; 5:225.-   22. Wang R Y-H, Shih, J W-k, Weiss S H, et al. Mycoplasma penetrans    infections in male homosexuals with AIDS: high seroprevalence and    association with Kaposi's sarcoma. Clin Infect Dis 1993; 17:724-29.-   23. Chang Y E, Cesarman E, Pessin M S, Lee F, Culpepper J, Knowles D    M, Moore P S. Identification of herpesvirus-like DNA sequences in    AIDS-associated Kaposi's sarcoma. Science 1994: 266; 1865-9.-   24. Moore P S, Cahng Y. Dectection of Herpes virus-like DNA    sequences in Kaposi's sarcoma inpatients with and those without HIV    infection. N Engl J Med 1995; 332:1181-5.-   25. Su I J, Hsu Y S, Chang Y C, Wang I W. Herpesvirus-like DNA    sequences in Kaposi's sarcoma from AIDS and non-AIDS pateints in    Taiwan. Lancet 1995; 345:722-3.-   26. Huang Y Q, Li J J, Kaplan M H, Polesz B, Katabira W C, Zhang D,    et al. Human herpesvirus-like nucleic acid in various forms of    Kaposi's sarcoma. Lancet 1995; 345:759-61.-   27. Dupin N, Grandadam M, Calvez V, Gorin I, Aubin J T, Harvard S,    et al, Herpesvirus-like DNA sequences in pateints with Mediterranean    Kaposi's sarcoma. Lancet 1995; 345:761-2.-   28, Collandre H, Ferris S, Grau O, Montagnier L, Blanchard A,    Kaposi's sarcoma and new herpes virus. Lancet 1995; 345:1043.-   29. Boshoff C, Whitby D, Hatziioannu T, Fisher C, van der Walt J,    Hatzakis A, Weiss R Schultz T. Kaposi's sarcoma-associated herpes    virus in HIV-negative Kaposi's Sarcoma. Lancet 1995; 345:1043-4.-   30. Cesarman E, Chang Y, Moore P S, Said J W c Knowles D M. Kaposi's    sarcoma associated herpes virus-like DNA sequences in aIDS-related    body-cavity-based lymphomas. N Engl J Med 1995; 332:1186-91.-   31. Knowles, D. M., G. Inghirami, A. Ubriaco, and R.    Dalla-Favera. 1989. Molecular genetic analysis of three    AIDS-associated neoplasms of uncertain lineage demonstrate their    B-cell derivation and the possible pathogenetic role of the    Epstein-Barr virus. Blod 74:792-799.-   32. Lisitsyn, N., N. Lisitsyn, and M. Wigler. 1993. Cloning the    differences between two complex genomes. Science 259:946-951.-   33. Miller G, Lipman M, Release of infectious Epstein-Barr virus by    transformed marmoset leukocytes. Proc Nat Acad Sci USA 1973;    70:190-4.-   34. zur Hausen, H., F. J. O'Neill, U. K. Freese, and E.    Hecker. 1978. Persisting oncogenic herpesvirus induced by tumor    promoter TPA. Naturer 272:373-375.-   35. Rabson, M., L. Heston, and G, Miller. 1983. Identification of a    rare Epstein-Barr virus variant which enhances early antigen    expression in Raji cells. Proc. Natl. aCad. Sci. USA 80:2762-2766.-   36. Luka, J., B. Kallin, and G. Klien. 1979. Induction of the    Epstein-Barr virus (EBV) cycle in latently infected cells by    n-butyrate. Virology 94:228-231.-   37. Calendar A, Billaud M, Aubry J-P, Bauchereau J, Vuillaume M,    Lenoir G M. Epstein-Barr virus (EBV) induces expression of B-cell    activation markers on in vitro infection of EBV-negative B-lymphoma    cells. Proc nat Acad Sci USA 1987; 84; 8060-4.-   39. Twobin, H., Staehelin, T., Gordon, J. Electrophoeretic transfer    of proteins from polyacrylamide gels to nitrocellulose sheets:    procedure and some applications Proc Natl Acad Sci USA 1979; 76:    4350-4354.-   39. van Grunsven, W. M. J., E. C. van Heerde, H. J. W. de    Haard, W. J. M. Spaan, and J. Middledorp. 1993. Gene mapping and    expression of two immunodominant Epstein-Barr virus capsid    proteins. J. Virol. 67:390 B-3916.-   40. Frickhofen N, Abkowitz J L, Safford M, Berry M,    Antunez-de-Mayolo J, et al. Persistent B19 Parvovirus infection in    pateints infected with human immunodeficiency virus type 1 (HIV-1):    a treatable cause of anemia in ADIS. Ann Intern Med 1990;    113:926-33.-   41. Biggar R J, Goedert J J, Hoofnagle J: Accelerated loss of    antibody to hepatitis B surface antigen among immunodeficient    homosexual men infected with HIV. N Engl J Med 1987; 316:630-31.-   42. Ensoli B, Barillari G, Salahuddin S Z et al. Tat protein of    HIV-1 stimulates growth of cells derived from Kaposi's sarcoma    lesions of AIDS patients. Nature 1990; 345:84,-   43. Vogel J, Hinrichs S H, Reynolds R K, Luciw P A, Jay G. The HIV    tat gene induces dermal lesions resembling Kaposi's sarcoma in    transgenic mice. Nature 1994; 335:601-11.-   44. Miles S A, Rezai A R, Salazar-Gonzalez J F, Stevens R H, Logan D    M, Mistuyasu R T, Taga T, et al. AIDS Kaposi's sarcoma-derived cells    produce and respond to interleukin-6. Proc Nath Acad Sci USA 1990;    87: 4068-72.

1-42. (canceled)
 43. An antisense molecule capable of hybridizing to anisolated nucleic acid which uniquely defines a herpesvirus associatedwith Kaposi's sarcoma and which comprises at least 30 nucleotides of anucleic acid selected from the group consisting of SEQ ID Nos: 1-15. 44.The antisense molecule of claim 43, wherein the molecule is a DNA. 45.The antisense molecule of claim 43, wherein the molecule is a RNA.
 46. Atriplex oligonucleotide capable of hybridizing with a double strandedisolated nucleic acid which uniquely defines a herpesvirus associatedwith Kaposi's sarcoma and which comprises at least 30 nucleotides of anucleic acid selected from the group consisting of SEQ ID Nos: 1-15. 47.A transgenic nonhuman mammal which comprises at least a portion of anisolated nucleic acid which uniquely defines a herpesvirus associatedwith Kaposi's sarcoma and which comprises at least 30 nucleotides of anucleic acid selected from the group consisting of SEQ ID Nos: 1-15,introduced into the mammal at an embryonic stage.
 48. An antisensemolecule capable of hybridizing to an isolated nucleic acid which is atleast 30 nucleotides in length and has a sequence which uniquely definesa herpesvirus associated with Kaposi's sarcoma, which herpesvirus ispresent in and recoverable from the HBL-6 cell line (ATCC Accession No.CRL 11762).
 49. The antisense molecule of claim 48, wherein the moleculeis a DNA.
 50. The antisense molecule of claim 48, wherein the moleculeis a RNA.
 51. A triplex oligonucleotide capable of hybridizing with adouble isolated nucleic acid which is at least 30 nucleotides in lengthand has a sequence which uniquely defines a herpesvirus associated withKaposi's sarcoma, which herpesvirus is present in and recoverable fromthe HBL-6 cell line (ATCC Accession No. CRL 11762).
 52. A transgenicnonhuman mammal which comprises at least a portion of an isolatednucleic acid which is at least 30 nucleotides in length and has asequence which uniquely defines a herpesvirus associated with Kaposi'ssarcoma, which herpesvirus is present in and recoverable from the HBL-6cell line (ATCC Accession No. CRL 11762) introduced into the mammal atan embryonic stage.
 53. A method of prophylaxis or treatment forKaposi's sarcoma (KS) by administering to a subject at risk for KS, anantibody that binds to an isolated DNA virus associated with Kaposi'sSarcoma wherein the viral DNA: (a) encodes a thymidine kinase; and (b)hybridizes under conditions of high stringency with a nucleic acidselected from the group consisting of SEQ ID Nos: 1-15 in apharmaceutically acceptable carrier.
 54. A method of immunizing asubject against a disease caused by the herpesvirus associated withKaposi's sarcoma which comprises administering to the subject aneffective immunizing dose of the vaccine of an associated herpesvirusassociated with Kaposi's sarcoma comprising an isolated nucleic acidwhich uniquely defines a herpesvirus associated with Kaposi's sarcomaand which comprises at least 30 nucleotides of a nucleic acid selectedfrom the group consisting of SEQ ID Nos: 1-15.
 55. A method forpreventing the development or transmission of herpesvirus associatedKaposi's sarcoma in a subject by treating a subject with Kaposi'ssarcoma (KS) comprising administering to the subject having a humanherpesvirus-associated KS a pharmaceutically effective amount of anantiviral agent in a pharmaceutically acceptable carrier, wherein theagent is effective to prevent the development or transmission of anisolated DNA virus associated with Kaposi's Sarcoma wherein the viralDNA: (a) encodes a thymidine kinase; and (b) hybridizes under conditionsof high stringency with a nucleic acid selected from the groupconsisting of SEQ ID Nos: 1-15.
 56. An isolated Kaposi'ssarcoma-associated herpesvirus (KSHV) glycoprotein H (gH) polypeptide.57. The isolated polypeptide of claim 56, wherein the polypeptide islinked to a second polypeptide to form a fusion protein.
 58. The fusionprotein of claim 57, wherein the second polypeptide isbeta-galactosidase.
 59. An antibody which specifically binds to thepolypeptide of claim
 56. 60. The antibody of claim 59, wherein theantibody is polyclonal antibody.
 61. The antibody of claim 59, whereinthe antibody is a monoclonal antibody.
 62. An antisense molecule capableof hybridizing to an isolated nucleic acid molecule encoding Kaposi'ssarcoma-associated herpesvirus (KSHV) glycoprotein H (gH).
 63. Theantisense molecule of claim 62, wherein the molecule is a nucleic acidderivative.
 64. The antisense molecule of claim 62, wherein the moleculeis an RNA derivative.
 65. A triplex oligonucleotide capable ofhybridizing with an isolated nucleic acid molecule encoding Kaposi'ssarcoma-associated herpesvirus (KSHV) glycoprotein H (gH).
 66. A peptideconjugated to a carrier protein wherein the peptide is encoded by atleast 30 nucleotides and has a sequence which is encoded by, anduniquely defines, Kaposi's sarcoma herpesvirus (KSHV), (deposited inBHL-6 cells under ATCC Accession No. 11762), wherein the sequence of thepeptide is present within the amino acid sequence of any of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35.